Lipase variants and microcapsule compositions comprising such lipase variants

ABSTRACT

The present invention relates to variants of a parent lipase which has lipase activity and comprise one or more substitutions corresponding to G23S, D27N, A40I, F51I,L, E56R, D57N, V60E,K, K98I, N101D, R118F, G163S, T231R, N233R, Y220F, T244E, and P256T using SEQ ID NO: 2 for numbering. The present invention also relates to compositions or microcapsule compositions comprising a lipase variant of the invention and to liquid products comprising a microcapsule composition of the invention as well as the use of said microcapsule composition for stabilizing lipase variants of the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. 371 national application ofinternational application no. PCT/EP2018/075852 filed Sep. 25, 2018which claims priority or the benefit under 35 U.S.C. 119 of Europeanapplication nos. 17193514.1 and 18180386.7 filed Sep. 27, 2017 and Jun.28, 2018, the contents of which are fully incorporated herein byreference.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to lipase variants, polynucleotidesencoding the variants, as well as methods of producing said variants.The invention also relates to compositions and microcapsule compositionscomprising a lipase variant of the invention and liquid productscomprising a microcapsule composition of the invention.

Description of the Related Art

Lipases are important biocatalysts which have shown to be useful forvarious applications. Wild-type Thermomyces lanuginosus lipase (synonymHumicola lanuginosa) sold under the tradename LIPOLASE™ and variantsthereof have been commercialized as active ingredient in detergentcompositions for the removal of lipid stains by hydrolyzingtriglycerides to generate fatty acids.

Detergent, cleaning and/or fabric care compositions comprise activeingredients which interfere with the ability of lipases to remove lipidstains. Many known Thermomyces lanuginosus lipase variants with goodwash performance form odor-generating short-chain fatty acids duringwash and/or has a short storage stability.

Thus, there is still a need and desire for lipases with improved washperformance, reduced odor-generation and/or improved storagestability/longer shelf life/increased thermostability.

SUMMARY OF THE INVENTION

The present invention relates to variants of a parent lipase whichvariant has lipase activity, has at least 60%, but less than 100%sequence identity with SEQ ID NO: 2 and comprises one or more (e.g.several) substitutions at positions corresponding to G23S, D27N, A40I,F5I,L, E56R, D57N, V60E,K, K98I, N101D, R118F, G163S, Y220F, T231R,N233R, T244E, and P256T.

The present invention furthermore relates to compositions comprising alipase variant of the invention as well as the use thereof forhydrolyzing a lipid substrate. Furthermore, the invention relates topolynucleotides encoding the variants of the invention; nucleic acidconstructs, vectors, and host cells comprising the polynucleotides.

In one aspect, the invention relates to microcapsule compositions,wherein the membrane of the microcapsule is produced by cross-linking ofa polybranched polyamine having a molecular weight of more than 1 kDa,wherein the microcapsule comprising a lipase variant of the invention.

In a further aspect the invention relates to microcapsule compositions,comprising a lipase variant of the invention entrapped in a compartmentformed by a membrane, which membrane is produced by cross-linking of (a)a polybranched polyamine having a molecular weight of more than 800 Da,and (b) an aliphatic or aromatic amine having a molecular weight of lessthan 300 Da; wherein the weight ratio of (a)/(b) is in the range of 0.1to 1000.

Finally, the invention relates to liquid products comprising amicrocapsule composition of the invention.

Definitions

Lipase: The terms “lipase”, “lipase enzyme”, “lipolytic enzyme”, “lipidesterase”, “lipolytic polypeptide”, and “lipolytic protein” refers to anenzyme in class EC3.1.1 as defined by Enzyme Nomenclature. It may havelipase activity (triacylglycerol lipase, EC3.1.1.3), cutinase activity(EC3.1.1.74), sterol esterase activity (EC3.1.1.13) and/or wax-esterhydrolase activity (EC3.1.1.50). For purposes of the present invention,lipase activity is determined according to the procedure described inthe Examples. In one aspect, the variants of the present invention haveat least 20%, e.g., at least 25%, at least 30%, at least 35%, at least40%, at least 45%, at least 50%, at least 55%, at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, or 100% of the lipase activity of the polypeptide ofSEQ ID NO: 2.

Allelic variant: The term “allelic variant” means any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

cDNA: The term “cDNA” means a DNA molecule that can be prepared byreverse transcription from a mature, spliced, mRNA molecule obtainedfrom a eukaryotic or prokaryotic cell. cDNA lacks intron sequences thatmay be present in the corresponding genomic DNA. The initial, primaryRNA transcript is a precursor to mRNA that is processed through a seriesof steps, including splicing, before appearing as mature spliced mRNA.

Coding sequence: The term “coding sequence” means a polynucleotide,which directly specifies the amino acid sequence of a variant. Theboundaries of the coding sequence are generally determined by an openreading frame, which begins with a start codon such as ATG, GTG or TTGand ends with a stop codon such as TAA, TAG, or TGA. The coding sequencemay be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.

Control sequences: The term “control sequences” means nucleic acidsequences necessary for expression of a polynucleotide encoding avariant of the present invention. Each control sequence may be native(i.e., from the same gene) or foreign (i.e., from a different gene) tothe polynucleotide encoding the variant or native or foreign to eachother. Such control sequences include, but are not limited to, a leader,polyadenylation sequence, propeptide sequence, promoter, signal peptidesequence, and transcription terminator. At a minimum, the controlsequences include a promoter, and transcriptional and translational stopsignals. The control sequences may be provided with linkers for thepurpose of introducing specific restriction sites facilitating ligationof the control sequences with the coding region of the polynucleotideencoding a variant.

Expression: The term “expression” includes any step involved in theproduction of a variant including, but not limited to, transcription,post-transcriptional modification, translation, post-translationalmodification, and secretion.

Expression vector: The term “expression vector” means a linear orcircular DNA molecule that comprises a polynucleotide encoding a variantand is operably linked to control sequences that provide for itsexpression.

Fragment: The term “fragment” means a polypeptide having one or more(e.g., several) amino acids absent from the amino and/or carboxylterminus of a polypeptide; wherein the fragment has lipase activity. Inone aspect, a fragment contains at least 50%, at least 55%, at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, or at least 95% but less than 100% of the number ofamino acids 1 to 269 of SEQ ID NO: 2.

High stringency conditions: The term “high stringency conditions” meansfor probes of at least 100 nucleotides in length, prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and 50% formamide, following standardSouthern blotting procedures for 12 to 24 hours. The carrier material isfinally washed three times each for 15 minutes using 2×SSC, 0.2% SDS at65° C.

Host cell: The term “host cell” means any cell type that is susceptibleto transformation, transfection, transduction, or the like with anucleic acid construct or expression vector comprising a polynucleotideof the present invention. The term “host cell” encompasses any progenyof a parent cell that is not identical to the parent cell due tomutations that occur during replication.

Improved property: The term “improved property” means a characteristicassociated with a variant that is improved compared to the parentlipase. Such improved properties include, but are not limited to,detergent stability, stability in detergent with protease present,protease stability, chemical stability, oxidation stability, pHstability, stability under storage conditions, and thermostability.

Isolated: The term “isolated” means a substance in a form or environmentwhich does not occur in nature. Non-limiting examples of isolatedsubstances include (1) any non-naturally occurring substance, (2) anysubstance including, but not limited to, any enzyme, variant, nucleicacid, protein, peptide or cofactor, that is at least partially removedfrom one or more or all of the naturally occurring constituents withwhich it is associated in nature; (3) any substance modified by the handof man relative to that substance found in nature; or (4) any substancemodified by increasing the amount of the substance relative to othercomponents with which it is naturally associated (e.g., multiple copiesof a gene encoding the substance; use of a stronger promoter than thepromoter naturally associated with the gene encoding the substance). Anisolated substance may be present in a fermentation broth sample.

Low stringency conditions: The term “low stringency conditions” meansfor probes of at least 100 nucleotides in length, prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and 25% formamide, following standardSouthern blotting procedures for 12 to 24 hours. The carrier material isfinally washed three times each for 15 minutes using 2×SSC, 0.2% SDS at50° C.

Mature polypeptide: The term “mature polypeptide” means a polypeptide inits final form following translation and any post-translationalmodifications, such as N-terminal processing, C-terminal truncation,glycosylation, phosphorylation, etc. In one aspect, the maturepolypeptide is amino acids 1 to 269 of SEQ ID NO: 2. It is known in theart that a host cell may produce a mixture of two or more differentmature polypeptides (i.e., with a different C-terminal and/or N-terminalamino acid) expressed by the same polynucleotide.

Mature polypeptide coding sequence: The term “mature polypeptide codingsequence” means a polynucleotide that encodes a mature polypeptidehaving lipase activity. In one aspect, the mature polypeptide codingsequence is nucleotides 1 to 807 of SEQ ID NO: 1.

Medium stringency conditions: The term “medium stringency conditions”means for probes of at least 100 nucleotides in length, prehybridizationand hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/mlsheared and denatured salmon sperm DNA, and 35% formamide, followingstandard Southern blotting procedures for 12 to 24 hours. The carriermaterial is finally washed three times each for 15 minutes using 2×SSC,0.2% SDS at 55° C.

Medium-high stringency conditions: The term “medium-high stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 35% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using2×SSC, 0.2% SDS at 60° C.

Mutant: The term “mutant” means a polynucleotide encoding a variant.

Nucleic acid construct: The term “nucleic acid construct” means anucleic acid molecule, either single- or double-stranded, which isisolated from a naturally occurring gene or is modified to containsegments of nucleic acids in a manner that would not otherwise exist innature or which is synthetic, which comprises one or more controlsequences.

Operably linked: The term “operably linked” means a configuration inwhich a control sequence is placed at an appropriate position relativeto the coding sequence of a polynucleotide such that the controlsequence directs expression of the coding sequence.

Parent or parent lipase: The term “parent” or “parent lipase” means alipase to which an alteration is made to produce the enzyme variants ofthe present invention. The parent lipase may be a naturally occurring(wild-type) polypeptide or a variant or fragment thereof.

Sequence identity: The relatedness between two amino acid sequences orbetween two nucleotide sequences is described by the parameter “sequenceidentity”.

For purposes of the present invention, the sequence identity between twoamino acid sequences is determined using the Needleman-Wunsch algorithm(Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implementedin the Needle program of the EMBOSS package (EMBOSS: The EuropeanMolecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16: 276-277), preferably version 5.0.0 or later. The parameters used aregap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62(EMBOSS version of BLOSUM62) substitution matrix. The output of Needlelabeled “longest identity” (obtained using the—nobrief option) is usedas the percent identity and is calculated as follows:(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present invention, the sequence identity between twodeoxyribonucleotide sequences is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, supra) as implemented in theNeedle program of the EMBOSS package (EMBOSS: The European MolecularBiology Open Software Suite, Rice et al., 2000, supra), preferablyversion 5.0.0 or later. The parameters used are gap open penalty of 10,gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBINUC4.4) substitution matrix. The output of Needle labeled “longestidentity” (obtained using the—nobrief option) is used as the percentidentity and is calculated as follows:(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment).

Stability: The stability of a lipase variant of the invention may beexpressed as the residual activity or the residual performance of saidlipase during or after exposure to various test conditions such as e.g.storage in a detergent composition, at various temperatures, at variouspH, in the presence of different components such as protease, chemicals,and/or oxidative substances (stress conditions) or during use in a washprocess. The stability of a lipase variant can be measured relative to aknown activity or performance of a parent lipase, e.g., the lipase shownas SEQ ID NO: 2, or alternatively to a known activity or performance ofthe lipase variant when initially added to a detergent compositionoptionally stored cold or frozen or relative to the lipase variantstored cold or frozen (unstressed conditions).

Subsequence: The term “subsequence” means a polynucleotide having one ormore (e.g., several) nucleotides absent from the 5′ and/or 3′ end of amature polypeptide coding sequence; wherein the subsequence encodes afragment having lipase activity. In one aspect, a subsequence containsat least 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, or at least 95% butless than 100% of the number of nucleotides 1 to 807 of SEQ ID NO: 1.

Variant: The term “variant” means a polypeptide having lipase activitycomprising an alteration, i.e., a substitution, insertion, and/ordeletion, at one or more (e.g., several) positions. A substitution meansreplacement of the amino acid occupying a position with a differentamino acid; a deletion means removal of the amino acid occupying aposition; and an insertion means adding an amino acid adjacent to andimmediately following the amino acid occupying a position. The variantsof the present invention have at least 20%, e.g., at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, or at least 100% of the lipase activity of the polypeptide of SEQID NO: 2.

Very high stringency conditions: The term “very high stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using2×SSC, 0.2% SDS at 70° C.

Very low stringency conditions: The term “very low stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 25% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using2×SSC, 0.2% SDS at 45° C.

Wild-type lipase: The term “wild-type” lipase means a lipase expressedby a naturally occurring microorganism, such as a bacterium, yeast, orfilamentous fungus found in nature.

Conventions for Designation of Variants

For purposes of the present invention, the polypeptide disclosed as SEQID NO: 2 is used to determine the corresponding amino acid residue inanother lipase. The amino acid sequence of another lipase is alignedwith SEQ ID NO: 2, and based on the alignment, the amino acid positionnumber corresponding to any amino acid residue in the polypeptidedisclosed in SEQ ID NO: 2 is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) asimplemented in the Needle program of the EMBOSS package (EMBOSS: TheEuropean Molecular Biology Open Software Suite, Rice et al., 2000,Trends Genet. 16: 276-277), preferably version 5.0.0 or later. Theparameters used are gap open penalty of 10, gap extension penalty of0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.

Identification of the corresponding amino acid residue in another lipasecan be determined by an alignment of multiple polypeptide sequencesusing several computer programs including, but not limited to, MUSCLE(multiple sequence comparison by log-expectation; version 3.5 or later;Edgar, 2004, Nucleic Acids Research 32: 1792-1797), MAFFT (version 6.857or later; Katoh and Kuma, 2002, Nucleic Acids Research 30: 3059-3066;Katoh et al., 2005, Nucleic Acids Research 33: 511-518; Katoh and Toh,2007, Bioinformatics 23: 372-374; Katoh et al., 2009, Methods inMolecular Biology 53709-64; Katoh and Toh, 2010, Bioinformatics26:1899-1900), and EMBOSS EMMA employing ClustalW (1.83 or later;Thompson et al., 1994, Nucleic Acids Research 22: 4673-4680), usingtheir respective default parameters.

When the other enzyme has diverged from the polypeptide of SEQ ID NO: 2such that traditional sequence-based comparison fails to detect theirrelationship (Lindahl and Elofsson, 2000, J. Mol. Biol. 295: 613-615),other pairwise sequence comparison algorithms can be used. Greatersensitivity in sequence-based searching can be attained using searchprograms that utilize probabilistic representations of polypeptidefamilies (profiles) to search databases. For example, the PSI-BLASTprogram generates profiles through an iterative database search processand is capable of detecting remote homologs (Atschul et al., 1997,Nucleic Acids Res. 25: 3389-3402). Even greater sensitivity can beachieved if the family or superfamily for the polypeptide has one ormore representatives in the protein structure databases. Programs suchas GenTHREADER (Jones, 1999, J. Mol. Biol. 287: 797-815; McGuffin andJones, 2003, Bioinformatics 19: 874-881) utilize information from avariety of sources (PSI-BLAST, secondary structure prediction,structural alignment profiles, and solvation potentials) as input to aneural network that predicts the structural fold for a query sequence.Similarly, the method of Gough et al., 2000, J. Mol. Biol. 313: 903-919,can be used to align a sequence of unknown structure with thesuperfamily models present in the SCOP database. These alignments can inturn be used to generate homology models for the polypeptide, and suchmodels can be assessed for accuracy using a variety of tools developedfor that purpose.

For proteins of known structure, several tools and resources areavailable for retrieving and generating structural alignments. Forexample the SCOP superfamilies of proteins have been structurallyaligned, and those alignments are accessible and downloadable. Two ormore protein structures can be aligned using a variety of algorithmssuch as the distance alignment matrix (Holm and Sander, 1998, Proteins33: 88-96) or combinatorial extension (Shindyalov and Bourne, 1998,Protein Engineering 11: 739-747), and implementation of these algorithmscan additionally be utilized to query structure databases with astructure of interest in order to discover possible structural homologs(e.g., Holm and Park, 2000, Bioinformatics 16: 566-567).

In describing the variants of the present invention, the nomenclaturedescribed below is adapted for ease of reference. The accepted IUPACsingle letter or three letter amino acid abbreviation is employed.

Substitutions. For an amino acid substitution, the followingnomenclature is used: Original amino acid, position, substituted aminoacid. Accordingly, the substitution of threonine at position 226 withalanine is designated as “Thr226Ala” or “T226A”. Multiple mutations areseparated by addition marks (“+”), e.g., “Gly205Arg+Ser411Phe” or“G205R+S411F”, representing substitutions at positions 205 and 411 ofglycine (G) with arginine (R) and serine (S) with phenylalanine (F),respectively.

Deletions. For an amino acid deletion, the following nomenclature isused: Original amino acid, position, *. Accordingly, the deletion ofglycine at position 195 is designated as “Gly195*” or “G195*”. Multipledeletions are separated by addition marks (“+”), e.g., “Gly195*+Ser411*”or “G195*+S411*”.

Insertions. For an amino acid insertion, the following nomenclature isused: Original amino acid, position, original amino acid, inserted aminoacid. Accordingly the insertion of lysine after glycine at position 195is designated “Gly195GlyLys” or “G195GK”. An insertion of multiple aminoacids is designated [Original amino acid, position, original amino acid,inserted amino acid #1, inserted amino acid #2; etc.]. For example, theinsertion of lysine and alanine after glycine at position 195 isindicated as “Gly195GlyLysAla” or “G195GKA”.

In such cases the inserted amino acid residue(s) are numbered by theaddition of lower case letters to the position number of the amino acidresidue preceding the inserted amino acid residue(s). In the aboveexample, the sequence would thus be:

Parent: Variant: 195 195 195a 195b G G - K - A

Multiple alterations. Variants comprising multiple alterations areseparated by addition marks (“+”), e.g., “Arg170Tyr+Gly195Glu” or“R170Y+G195E” representing a substitution of arginine and glycine atpositions 170 and 195 with tyrosine and glutamic acid, respectively.

Different alterations. Where different alterations can be introduced ata position, the different alterations are separated by a comma, e.g.,“Arg170Tyr,Glu” or “R170Y,E” represents a substitution of arginine atposition 170 with tyrosine or glutamic acid. Thus,“Tyr167Gly,Ala+Arg170Gly,Ala” designates the following variants:

“Tyr167Gly+Arg170Gly”, “Tyr167Gly+Arg170Ala”, “Tyr167Ala+Arg170Gly”, and“Tyr167Ala+Arg170Ala”.

DETAILED DESCRIPTION OF THE INVENTION

Disclosed are variants of a parent lipase which variants have lipaseactivity, has at least 60%, but less than 100% sequence identity withSEQ ID NO: 2, e.g., derived from Thermomyces lanuginosus.

Variants

The present invention provides a variant of a parent lipase whichvariant has lipase activity, has at least 60%, but less than 100%sequence identity with SEQ ID NO: 2 and comprises one or more (e.g.several) substitutions at positions corresponding to G23S, D27N, A40I,F51I,L, E56R, D57N, V60E,K, K98I, N101D, R118F, G163S, Y220F, T231R,N233R, T244E, and P256T.

Preferably the lipase variant has at least 60%, but less than 100%sequence identity with SEQ ID NO: 2 and comprises one or moresubstitutions at positions corresponding to G23S, D27N, A40I, F51I,L,E56R, D57N, V60E,K, K98I, N101D, R118F, G163S, Y220F, T244E, and P256T.Preferably the variant additionally comprises one or both substitutionsat positions corresponding to T231R and/or N233R.

A preferred variant comprises a substitution at a position correspondingto G23S. A preferred variant comprises a substitution at a positioncorresponding to D27N. A preferred variant comprises a substitution at aposition corresponding to A40I. A preferred variant comprises asubstitution at a position corresponding to F51I,L. A preferred variantcomprises a substitution at a position corresponding to E56R. Apreferred variant comprises a substitution at a position correspondingto D57N. A preferred variant comprises a substitution at a positioncorresponding to V60E,K. A preferred variant comprises a substitution ata position corresponding to K981. A preferred variant comprises asubstitution at a position corresponding to N101D. A preferred variantcomprises a substitution at a position corresponding to R118F. Apreferred variant comprises a substitution at a position correspondingto G163S. A preferred variant comprises a substitution at a positioncorresponding to Y220F. A preferred variant comprises a substitution ata position corresponding to T244E. A preferred variant comprises asubstitution at a position corresponding to P256T.

A preferred variant comprises one or more substitutions at positionscorresponding to F51I,L, E56R and/or R118F.

In preferred embodiment, a variant of the invention comprises any one ofthe following set of substitutions:

G23S + T231R + N233R D27N + T231R + N233R A40I + T231R + N233R F51I +T231R + N233R F51L + T231R + N233R E56R + T231R + N233R D57N + T231R +N233R V60E + T231R + N233R V60K + T231R + N233R K98I + T231R + N233RN101D + T231R + N233R R118F + T231R + N233R G163S + T231R + N233RY220F + T231R + N233R T231R + N233R + T244E T231R + N233R + P256T

In an embodiment the variant comprises substitutions at positionscorresponding to T231R+N233R and one or more (e.g., several)substitutions at positions corresponding to G23S, D27N, A40I, F51I,L,E56R, D57N, V60E,K, K98I, N101D, R118F, G163S, Y220F, T244E, and P256T.

In a preferred embodiment the variant comprises substitutionscorresponding to E56R+T231R+N233R and one or more (e.g., several)substitutions at positions corresponding to G23S, D27N, A40I, F51I,L,D57N, V60E,K, K98I, N101D, R118F, G163S, Y220F, T244E, and P256T.

In a preferred embodiment the variant comprises substitutions atpositions corresponding to R118F+T231R+N233R and one or more (e.g.,several) substitutions at positions corresponding to G23S, D27N, A40I,F51I,L, E56R, D57N, V60E,K, K98I, N101D, G163S, Y220F, T244E, and P256T.

In a more preferred embodiment variant comprises substitutions atpositions corresponding to E56R+R118F+T231R+N233R and one or more (e.g.,several) substitutions at positions corresponding to G23S, D27N, A40I,F51I,L, D57N, V60E,K, K98I, N101D, G163S, Y220F, T244E, and P256T.

In an even more preferred embodiment the variant comprises substitutionsat positions corresponding to E56R+R118F+T231R+N233R+P256T and one ormore (e.g., several) substitutions at positions corresponding to G23S,D27N, A40I, F51I,L, D57N, V60E,K, K98I, N101D, G163S, Y220F, and T244E.

In an even more preferred embodiment the variant comprises substitutionsat positions corresponding to F51I,L+E56R+R118F+T231R+N233R and one ormore (e.g., several) substitutions at positions corresponding to G23S,D27N, A40I, D57N, V60E,K, K98I, N101D, G163S, Y220F, T244E and P256T.

In an even more preferred embodiment the variant comprises substitutionsat positions corresponding to F51I,L+E56R+R118F+T231R+N233R+P256T andone or more (e.g., several) substitutions at positions corresponding toG23S, D27N, A40I, D57N, V60E,K, K98I, N101D, G163S, Y220F, and T244E.

In another more preferred embodiment the variant comprises substitutionsat positions corresponding to G23S+F51I,L+E56R+R118F+T231R+N233R and oneor more (e.g., several) substitutions at positions corresponding toD27N, A40I, D57N, V60E,K, K98I, N101D, G163S, Y220F, T244E, and P256T.

In a further even more preferred embodiment the variant comprisessubstitutions at positions corresponding toD27N+F51I,L+E56R+R118F+T231R+N233R+P256T and one or more (e.g., several)substitutions at positions corresponding to G23S, A40I, D57N, V60E,K,K98I, N101D, G163S, Y220F, and T244E.

In an additional even more preferred embodiment variant comprisessubstitutions at positions corresponding toA40I+F51I,L+E56R+R118F+T231R+N233R and one or more (e.g., several)substitutions at positions corresponding to G23S, D27N, D57N, V60E,K,K98I, N101D, G163S, Y220F, T244E and P256T.

In a further even more preferred embodiment the variant comprisessubstitutions at positions corresponding toD27N+F51I,L+E56R+R118F+T231R+N233R+P256T and one or more (e.g., several)substitutions at positions corresponding to G23S, A40I, D57N, V60E,K,K98I, N101D, G163S, Y220F, and T244E.

In an additional even more preferred embodiment variant comprisessubstitutions at positions corresponding toA40I+F51I,L+E56R+R118F+T231R+N233R+P256T and one or more (e.g., several)substitutions at positions corresponding to G23S, D27N, D57N, V60E,K,K98I, N101D, G163S, Y220F, and T244E.

In a further even more preferred embodiment the variant comprisessubstitutions at positions corresponding toF51I,L+E56R+D57N+R118F+T231R+N233R+P256T and one or more (e.g., several)substitutions at positions corresponding to G23S, D27N, A40I, V60E,K,K98I, N101D, G163S, Y220F, and T244E.

In a further preferred embodiments the variant comprises substitutionsat positions corresponding toF51I,L+E56R+D57N+K98I+R118F+T231R+N233R+P256T and one or more (e.g.,several) substitutions at positions corresponding to G23S, D27N, A40I,V60E,K, N101D, G163S, Y220F, and T244E. In a further more preferredembodiment the variant comprises substitutions at positionscorresponding to F51I,L+E56R+D57N+K98I+R118F+G163S+T231R+N233R+P256T andone or more (e.g., several) substitutions at positions corresponding toG23S, D27N, A40I, V60EK, N101D, Y220F, and T244E.

In a further even more preferred embodiment the variant comprisessubstitutions at positions corresponding toF51I,L+E56R+D57N+K98I+R118F+G163S+T231R+N233R+T244E+P256T and one ormore (e.g., several) substitutions at positions corresponding to G23S,D27N, A40I, V60E,K, N101D and Y220F.

In a further preferred embodiments the variant comprises substitutionsat positions corresponding toF51I,L+E56R+D57N+V60E,K+K98I+R118F+T231R+N233R+P256T and one or more(e.g., several) substitutions at positions corresponding to G23S, D27N,A40I, N101D, G163S, Y220F, and T244E.

In a further preferred embodiments the variant comprises substitutionsat positions corresponding toF51I,L+E56R+D57N+V60E,K+K98I+N101D+R118F+T231R+N233R+P256T and one ormore (e.g., several) substitutions at positions corresponding to G23S,D27N, A40I, G163S, Y220F, and T244E. In a further preferred embodimentsthe variant comprises substitutions at positions corresponding toF51I,L+E56R+D57N+N101D+K98I+R118F+T231R+N233R+P256T and one or more(e.g., several) substitutions at positions corresponding to G23S, D27N,A40I, V60E,K, N101D, G163S, Y220F, and T244E.

Particularly preferred embodiments include variants comprisingsubstitutions at positions corresponding to one of the following set ofsubstitutions:

-   R118F+T231R+N233R+P256T;-   A40I+R118F+T231R+N233R;-   F51I+E56R+R118F+T231R+N233R;-   F51L+E56R+R118F+T231R+N233R;-   E56R+D57N+R118F+T231R+N233R;-   E56R+V60K+R118F+T231R+N233R;-   G23S+E56R+R118F+T231R+N233R;-   D27N+E56R+R118F+T231R+N233R;-   F51I+E56R+R118F+T231R+N233R;-   E56R+R118F+T231R+N233R+P256T;-   G23S+D27N+E56R+R118F+T231R+N233R;-   G23S+F51I+E56R+R118F+T231R+N233R;-   G23S+E56R+R118F+T231R+N233R+P256T;-   D27N+F51I+E56R+R118F+T231R+N233R;-   D27N+E56R+R118F+T231R+N233R+P256T;-   F51I+E56R+R118F+T231R+N233R+P256T;-   G23S+D27N+F51I+E56R+R118F+T231R+N233R;-   G23S+D27N+E56R+R118F+T231R+N233R+P256T;-   G23S+D27N+F51I+E56R+V60K+R118F+T231R+N233R+P256T;-   G23S+D27N+F51I+E56R+V60E+R118F+T231R+N233R+P256T;-   G23S+F51I+E56R+R118F+T231R+N233R+P256T;-   D27N+F51I+E56R+R118F+T231R+N233R+P256T;-   G23S+D27N+F51I+E56R+R118F+T231R+N233R+P256T;-   A40I+E56R+R118F+T231R+N233R;-   F51L+E56R+R118F+T231R+N233R;-   D57N+E56R+R118F+T231R+N233R;-   K98I+E56R+R118F+T231R+N233R;-   G163S+E56R+R118F+T231R+N233R;-   A40I+F51L+E56R+R118F+T231R+N233R;-   A40I+D57N+E56R+R118F+T231R+N233R;-   A40I+K98I+E56R+R118F+T231R+N233R;-   A40I+G163S+E56R+R118F+T231R+N233R;-   A40I+E56R+R118F+T231R+N233R+P256T;-   F51L+D57N+E56R+R118F+T231R+N233R;-   F51L+K98I+E56R+R118F+T231R+N233R;-   F51L+G163S+E56R+R118F+T231R+N233R;-   F51L+E56R+R118F+T231R+N233R+P256T;-   D57N+K98I+E56R+R118F+T231R+N233R;-   D57N+G163S+E56R+R118F+T231R+N233R;-   D57N+E56R+R118F+T231R+N233R+P256T;-   K98I+G163S+E56R+R118F+T231R+N233R;-   K98I+E56R+R118F+T231R+N233R+P256T;-   G163S+E56R+R118F+T231R+N233R+P256T;-   A40I+F51L+D57N+E56R+R118F+T231R+N233R;-   A40I+F51L+K98I+E56R+R118F+T231R+N233R;-   A40I+F51L+G163S+E56R+R118F+T231R+N233R;-   A40I+F51L+E56R+R118F+T231R+N233R+P256T;-   A40I+D57N+K98I+E56R+R118F+T231R+N233R;-   A40I+D57N+G163S+E56R+R118F+T231R+N233R;-   A40I+D57N+E56R+R118F+T231R+N233R+P256T;-   A40I+K98I+G163S+E56R+R118F+T231R+N233R;-   A40I+K98I+E56R+R118F+T231R+N233R+P256T;-   A40I+G163S+E56R+R118F+T231R+N233R+P256T;-   F51L+D57N+K98I+E56R+R118F+T231R+N233R;-   F51L+D57N+G163S+E56R+R118F+T231R+N233R;-   F51L+D57N+E56R+R118F+T231R+N233R+P256T;-   F51L+K98I+G163S+E56R+R118F+T231R+N233R;-   F51L+K98I+E56R+R118F+T231R+N233R+P256T;-   F51L+G163S+E56R+R118F+T231R+N233R+P256T;-   D57N+K98I+G163S+E56R+R118F+T231R+N233R;-   D57N+K98I+E56R+R118F+T231R+N233R+P256T;-   D57N+G163S+E56R+R118F+T231R+N233R+P256T;-   K98I+G163S+E56R+R118F+T231R+N233R+P256T;-   A40I+F51L+D57N+K98I+E56R+R118F+T231R+N233R;-   A40I+F51L+D57N+G163S+E56R+R118F+T231R+N233R;-   A40I+F51L+D57N+E56R+R118F+T231R+N233R+P256T;-   A40I+F51L+K98I+G163S+E56R+R118F+T231R+N233R;-   A40I+F51L+K98I+E56R+R118F+T231R+N233R+P256T;-   A40I+F51L+G163S+E56R+R118F+T231R+N233R+P256T;-   A40I+D57N+K98I+G163S+E56R+R118F+T231R+N233R;-   A40I+D57N+K98I+E56R+R118F+T231R+N233R+P256T;-   A40I+D57N+G163S+E56R+R118F+T231R+N233R+P256T;-   A40I+K98I+G163S+E56R+R118F+T231R+N233R+P256T;-   F51L+D57N+K98I+G163S+E56R+R118F+T231R+N233R;-   F51L+D57N+K98I+E56R+R118F+T231R+N233R+P256T;-   F51L+D57N+G163S+E56R+R118F+T231R+N233R+P256T;-   F51L+K98I+G163S+E56R+R118F+T231R+N233R+P256T;-   D57N+K98I+G163S+E56R+R118F+T231R+N233R+P256T;-   A40I+F51L+D57N+K98I+G163S+E56R+R118F+T231R+N233R;-   A40I+F51L+D57N+K98I+E56R+R118F+T231R+N233R+P256T;-   A40I+F51L+D57N+G163S+E56R+R118F+T231R+N233R+P256T;-   A40I+F51L+K98I+G163S+E56R+R118F+T231R+N233R+P256T;-   A40I+D57N+K98I+G163S+E56R+R118F+T231R+N233R+P256T;-   F51L+D57N+K98I+G163S+E56R+R118F+T231R+N233R+P256T;-   A40I+F51L+D57N+K98I+G163S+E56R+R118F+T231R+N233R+P256T;-   A40I+F51L+E56R+D57N+K98I+R118F+G163S+T231R+N233R+P256T;-   A40I+E56R+R118F+T231R+N233R;-   E56R+R118F+T231R+N233R+T244E;-   G23S+D27N+E56R+R118F+T231R+N233R;-   G23S+A40I+E56R+R118F+T231R+N233R;-   G23S+F51I+E56R+R118F+T231R+N233R;-   G23S+E56R+R118F+T231R+N233R+T244E;-   G23S+E56R+R118F+T231R+N233R+P256T;-   D27N+A40I+E56R+R118F+T231R+N233R;-   D27N+F51I+E56R+R118F+T231R+N233R;-   D27N+E56R+R118F+T231R+N233R+T244E;-   D27N+E56R+R118F+T231R+N233R+P256T;-   A40I+F51I+E56R+R118F+T231R+N233R;-   A40I+E56R+R118F+T231R+N233R+T244E;-   A40I+E56R+R118F+T231R+N233R+P256T;-   F51I+E56R+R118F+T231R+N233R+T244E;-   F51I+E56R+R118F+T231R+N233R+P256T;-   E56R+R118F+T231R+N233R+T244E+P256T;-   G23S+D27N+A40I+E56R+R118F+T231R+N233R;-   G23S+D27N+A40I+E56R+V60K+R118F+T231R+N233R;-   G23S+D27N+A40I+E56R+V60E+R118F+T231R+N233R;-   G23S+D27N+F51I+E56R+R118F+T231R+N233R;-   G23S+D27N+E56R+R118F+T231R+N233R+T244E;-   G23S+D27N+E56R+R118F+T231R+N233R+P256T;-   G23S+A40I+F51I+E56R+R118F+T231R+N233R;-   G23S+A40I+E56R+R118F+T231R+N233R+T244E;-   G23S+A40I+E56R+R118F+T231R+N233R+P256T;-   G23S+F51I+E56R+R118F+T231R+N233R+T244E;-   G23S+F51I+E56R+R118F+T231R+N233R+P256T;-   G23S+E56R+R118F+T231R+N233R+T244E+P256T;-   G23S+E56R+V60K+R118F+T231R+N233R+T244E+P256T;-   G23S+E56R+V60E+R118F+T231R+N233R+T244E+P256T;-   D27N+A40I+F51I+E56R+R118F+T231R+N233R;-   D27N+A40I+E56R+R118F+T231R+N233R+T244E;-   D27N+A40I+E56R+R118F+T231R+N233R+P256T;-   D27N+F51I+E56R+R118F+T231R+N233R+T244E;-   D27N+F51I+E56R+R118F+T231R+N233R+P256T;-   D27N+E56R+R118F+T231R+N233R+T244E+P256T;-   A40I+F51I+E56R+R118F+T231R+N233R+T244E;-   A40I+F51I+E56R+R118F+T231R+N233R+P256T;-   A40I+E56R+R118F+T231R+N233R+T244E+P256T;-   F51I+E56R+R118F+T231R+N233R+T244E+P256T;-   F51I+E56R+V60K+R118F+T231R+N233R+T244E+P256T;-   F51I+E56R+V60E+R118F+T231R+N233R+T244E+P256T;-   G23S+D27N+A40I+F51I+E56R+R118F+T231R+N233R;-   G23S+D27N+A40I+F51I+E56R+V60K+R118F+T231R+N233R;-   G23S+D27N+A40I+F51I+E56R+V60E+R118F+T231R+N233R;-   G23S+D27N+A40I+E56R+R118F+T231R+N233R+T244E;-   G23S+D27N+A40I+E56R+V60K+R118F+T231R+N233R+T244E:-   G23S+D27N+A40I+E56R+V60E+R118F+T231R+N233R+T244E;-   G23S+D27N+A40I+E56R+R118F+T231R+N233R+P256T;-   G23S+D27N+F51I+E56R+R118F+T231R+N233R+T244E;-   G23S+D27N+F51I+E56R+R118F+T231R+N233R+P256T;-   G23S+D27N+E56R+R118F+T231R+N233R+T244E+P256T;-   G23S+A40I+F51I+E56R+R118F+T231R+N233R+T244E;-   G23S+A40I+F51I+E56R+R118F+T231R+N233R+P256T;-   G23S+A40I+E56R+R118F+T231R+N233R+T244E+P256T;-   G23S+F51I+E56R+R118F+T231R+N233R+T244E+P256T;-   D27N+A40I+F51I+E56R+R118F+T231R+N233R+T244E;-   D27N+A40I+F51I+E56R+R118F+T231R+N233R+P256T;-   D27N+A40I+E56R+R118F+T231R+N233R+T244E+P256T;-   D27N+F51I+E56R+R118F+T231R+N233R+T244E+P256T;-   A40I+F51I+E56R+R118F+T231R+N233R+T244E+P256T;-   A40I+F51I+E56R+V60K+R118F+T231R+N233R+T244E+P256T;-   A40I+F51I+E56R+V60E+R118F+T231R+N233R+T244E+P256T;-   G23S+D27N+A40I+F51I+E56R+R118F+T231R+N233R+T244E;-   G23S+D27N+A40I+F51I+E56R+V60K+R118F+T231R+N233R+T244E;-   G23S+D27N+A40I+F51I+E56R+R118F+T231R+N233R+P256T;-   G23S+D27N+A40I+F51I+E56R+V60K+R118F+T231R+N233R+P256T;-   G23S+D27N+A40I+F51I+E56R+V60E+R118F+T231R+N233R+P256T;-   G23S+D27N+A40I+E56R+R118F+T231R+N233R+T244E+P256T;-   G23S+D27N+F51I+E56R+R118F+T231R+N233R+T244E+P256T;-   G23S+A40I+F51I+E56R+R118F+T231R+N233R+T244E+P256T;-   D27N+A40I+F51I+E56R+R118F+T231R+N233R+T244E+P256T;-   D27N+A40I+F51I+E56R+V60K+R118F+T231R+N233R+T244E+P256T;-   D27N+A40I+F51I+E56R+V60E+R118F+T231R+N233R+T244E+P256T;-   G23S+D27N+A40I+F51I+E56R+R118F+T231R+N233R+T244E+P256T;-   G23S+D27N+A40I+F51I+E56R+K98I+N101D+R118F+T231R+N233R+T244E+P256T;-   G23S+D27N+A40I+F51I+E56R+V60K+R118F+T231R+N233R+T244E+P256T;-   G23S+D27N+A40I+F51I+E56R+V60E+R118F+T231R+N233R+T244E+P256T;-   F51I+E56R+R118F+T231R+N233R;-   E56R+R118F+T231R+N233R+T244E;-   D27N+F51I+E56R+R118F+T231R+N233R;-   D27N+E56R+R118F+T231R+N233R+T244E;-   F51I+E56R+R118F+T231R+N233R+T244E;-   D27N+F51I+E56R+R118F+T231R+N233R+T244E;-   G23S+E56R+R118F+T231R+N233R;-   D27N+E56R+R118F+T231R+N233R;-   K98I+E56R+R118F+T231R+N233R;-   Y220F+E56R+R118F+T231R+N233R;-   E56R+R118F+T231R+N233R+T244E;-   G23S+D27N+E56R+R118F+T231R+N233R;-   G23S+F51I+E56R+R118F+T231R+N233R;-   G23S+K98I+E56R+R118F+T231R+N233R;-   G23S+Y220F+E56R+R118F+T231R+N233R;-   G23S+E56R+R118F+T231R+N233R+T244E;-   G23S+E56R+R118F+T231R+N233R+P256T;-   D27N+F51I+E56R+R118F+T231R+N233R;-   D27N+K98I+E56R+R118F+T231R+N233R;-   D27N+Y220F+E56R+R118F+T231R+N233R;-   D27N+E56R+R118F+T231R+N233R+T244E;-   D27N+E56R+R118F+T231R+N233R+P256T;-   F51I+K98I+E56R+R118F+T231R+N233R;-   F51I+Y220F+E56R+R118F+T231R+N233R;-   F51I+E56R+R118F+T231R+N233R+T244E;-   F51I+E56R+R118F+T231R+N233R+P256T;-   K98I+Y220F+E56R+R118F+T231R+N233R;-   K98I+E56R+R118F+T231R+N233R+T244E;-   K98I+E56R+R118F+T231R+N233R+P256T;-   Y220F+E56R+R118F+T231R+N233R+T244E;-   Y220F+E56R+R118F+T231R+N233R+P256T;-   E56R+R118F+T231R+N233R+T244E+P256T;-   G23S+D27N+F51I+E56R+R118F+T231R+N233R;-   G23S+D27N+K98I+E56R+R118F+T231R+N233R;-   G23S+D27N+Y220F+E56R+R118F+T231R+N233R;-   G23S+D27N+E56R+R118F+T231R+N233R+T244E;-   G23S+D27N+E56R+R118F+T231R+N233R+P256T;-   G23S+F51I+K98I+E56R+R118F+T231R+N233R;-   G23S+F51I+Y220F+E56R+R118F+T231R+N233R;-   G23S+F51I+E56R+R118F+T231R+N233R+T244E;-   G23S+F51I+E56R+R118F+T231R+N233R+P256T;-   G23S+K98I+Y220F+E56R+R118F+T231R+N233R;-   G23S+K98I+E56R+R118F+T231R+N233R+T244E;-   G23S+K98I+E56R+R118F+T231R+N233R+P256T;-   G23S+Y220F+E56R+R118F+T231R+N233R+T244E;-   G23S+Y220F+E56R+R118F+T231R+N233R+P256T;-   G23S+E56R+R118F+T231R+N233R+T244E+P256T;-   D27N+F51I+K98I+E56R+R118F+T231R+N233R;-   D27N+F51I+Y220F+E56R+R118F+T231R+N233R;-   D27N+F51I+E56R+R118F+T231R+N233R+T244E;-   D27N+F51I+E56R+R118F+T231R+N233R+P256T;-   D27N+K98I+Y220F+E56R+R118F+T231R+N233R;-   D27N+K98I+E56R+R118F+T231R+N233R+T244E;-   D27N+K98I+E56R+R118F+T231R+N233R+P256T;-   D27N+Y220F+E56R+R118F+T231R+N233R+T244E;-   D27N+Y220F+E56R+R118F+T231R+N233R+P256T;-   D27N+E56R+R118F+T231R+N233R+T244E+P256T;-   F51I+K98I+Y220F+E56R+R118F+T231R+N233R;-   F51I+K98I+E56R+R118F+T231R+N233R+T244E;-   F51I+K98I+E56R+R118F+T231R+N233R+P256T;-   F51I+Y220F+E56R+R118F+T231R+N233R+T244E;-   F51I+Y220F+E56R+R118F+T231R+N233R+P256T;-   F51I+E56R+R118F+T231R+N233R+T244E+P256T;-   K98I+Y220F+E56R+R118F+T231R+N233R+T244E;-   K98I+Y220F+E56R+R118F+T231R+N233R+P256T;-   K98I+E56R+R118F+T231R+N233R+T244E+P256T;-   Y220F+E56R+R118F+T231R+N233R+T244E+P256T;-   G23S+D27N+F51I+K98I+E56R+R118F+T231R+N233R;-   G23S+D27N+F51I+Y220F+E56R+R118F+T231R+N233R;-   G23S+D27N+F51I+E56R+R118F+T231R+N233R+T244E;-   G23S+D27N+F51I+E56R+R118F+T231R+N233R+P256T;-   G23S+D27N+K98I+Y220F+E56R+R118F+T231R+N233R;-   G23S+D27N+K98I+E56R+R118F+T231R+N233R+T244E;-   G23S+D27N+K98I+E56R+R118F+T231R+N233R+P256T;-   G23S+D27N+Y220F+E56R+R118F+T231R+N233R+T244E;-   G23S+D27N+Y220F+E56R+R118F+T231R+N233R+P256T;-   G23S+D27N+E56R+R118F+T231R+N233R+T244E+P256T;-   G23S+F51I+K98I+Y220F+E56R+R118F+T231R+N233R;-   G23S+F51I+K98I+E56R+R118F+T231R+N233R+T244E;-   G23S+F51I+K98I+E56R+R118F+T231R+N233R+P256T;-   G23S+F51I+Y220F+E56R+R118F+T231R+N233R+T244E;-   G23S+F51I+Y220F+E56R+R118F+T231R+N233R+P256T;-   G23S+F51I+E56R+R118F+T231R+N233R+T244E+P256T;-   G23S+K98I+Y220F+E56R+R118F+T231R+N233R+T244E;-   G23S+K98I+Y220F+E56R+R118F+T231R+N233R+P256T;-   G23S+K98I+E56R+R118F+T231R+N233R+T244E+P256T;-   G23S+Y220F+E56R+R118F+T231R+N233R+T244E+P256T;-   D27N+F51I+K98I+Y220F+E56R+R118F+T231R+N233R;-   D27N+F51I+K98I+E56R+R118F+T231R+N233R+T244E;-   D27N+F51I+K98I+E56R+R118F+T231R+N233R+P256T;-   D27N+F51I+Y220F+E56R+R118F+T231R+N233R+T244E;-   D27N+F51I+Y220F+E56R+R118F+T231R+N233R+P256T;-   D27N+F51I+E56R+R118F+T231R+N233R+T244E+P256T;-   D27N+K98I+Y220F+E56R+R118F+T231R+N233R+T244E;-   D27N+K98I+Y220F+E56R+R118F+T231R+N233R+P256T;-   D27N+K98I+E56R+R118F+T231R+N233R+T244E+P256T;-   D27N+Y220F+E56R+R118F+T231R+N233R+T244E+P256T;-   F51I+K98I+Y220F+E56R+R118F+T231R+N233R+T244E;-   F51I+K98I+Y220F+E56R+R118F+T231R+N233R+P256T;-   F51I+K98I+E56R+R118F+T231R+N233R+T244E+P256T;-   F51I+Y220F+E56R+R118F+T231R+N233R+T244E+P256T;-   K98I+Y220F+E56R+R118F+T231R+N233R+T244E+P256T;-   G23S+D27N+F51I+K98I+Y220F+E56R+R118F+T231R+N233R;-   G23S+D27N+F51I+K98I+E56R+R118F+T231R+N233R+T244E;-   G23S+D27N+F51I+K98I+E56R+R118F+T231R+N233R+P256T;-   G23S+D27N+F51I+Y220F+E56R+R118F+T231R+N233R+T244E;-   G23S+D27N+F51I+Y220F+E56R+R118F+T231R+N233R+P256T;-   G23S+D27N+F51I+E56R+R118F+T231R+N233R+T244E+P256T;-   G23S+D27N+K98I+Y220F+E56R+R118F+T231R+N233R+T244E;-   G23S+D27N+K98I+Y220F+E56R+R118F+T231R+N233R+P256T;-   G23S+D27N+K98I+E56R+R118F+T231R+N233R+T244E+P256T;-   G23S+D27N+Y220F+E56R+R118F+T231R+N233R+T244E+P256T;-   G23S+F51I+K98I+Y220F+E56R+R118F+T231R+N233R+T244E;-   G23S+F51I+K98I+Y220F+E56R+R118F+T231R+N233R+P256T;-   G23S+F51I+K98I+E56R+R118F+T231R+N233R+T244E+P256T;-   G23S+F51I+Y220F+E56R+R118F+T231R+N233R+T244E+P256T;-   G23S+K98I+Y220F+E56R+R118F+T231R+N233R+T244E+P256T;-   D27N+F51I+K98I+Y220F+E56R+R118F+T231R+N233R+T244E;-   D27N+F51I+K98I+Y220F+E56R+R118F+T231R+N233R+P256T;-   D27N+F51I+K98I+E56R+R118F+T231R+N233R+T244E+P256T;-   D27N+F51I+Y220F+E56R+R118F+T231R+N233R+T244E+P256T;-   D27N+K98I+Y220F+E56R+R118F+T231R+N233R+T244E+P256T;-   F51I+K98I+Y220F+E56R+R118F+T231R+N233R+T244E+P256T;-   G23S+D27N+F51I+K98I+Y220F+E56R+R118F+T231R+N233R+T244E;-   G23S+D27N+F51I+K98I+Y220F+E56R+R118F+T231R+N233R+P256T;-   G23S+D27N+F51I+K98I+E56R+R118F+T231R+N233R+T244E+P256T;-   G23S+D27N+F51I+Y220F+E56R+R118F+T231R+N233R+T244E+P256T;-   G23S+D27N+K98I+Y220F+E56R+R118F+T231R+N233R+T244E+P256T;-   G23S+F51I+K98I+Y220F+E56R+R118F+T231R+N233R+T244E+P256T;-   D27N+F51I+K98I+Y220F+E56R+R118F+T231R+N233R+T244E+P256T;-   G23S+D27N+F51I+K98I+Y220F+E56R+R118F+T231R+N233R+T244E+P256T;-   G23S+D27N+F51I+E56R+K98I+R118F+Y220F+T231R+N233R+T244E+P256T.

A lipase variant of the invention has at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95% identity, at least 96%, at least 97%, at least 98%, or atleast 99%, but less than 100% sequence identity to the parent lipase.

In a preferred embodiment, a variant of the invention has at least 60%,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95% identity, at least 96%, at least 97%, at least98%, or at least 99%, but less than 100% sequence identity to SEQ ID NO:2.

A variant of the invention may have from 1-40, 1-30, 1-20, such as 1-12,such as 1-11, such as 1-10, such as 1-9, such as 1-8, such as 1-7, suchas 1-6, such as 1-5, such as 1-4, such as 1-3, or such as 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 substitutions.

A variant of the invention may in comparison to the parent lipase haveone or more of the following properties: improved wash performance,reduced odor generation, improved storage stability, longer shelf lifeand/or increased thermostability.

A lipase variant of the invention may further comprise one or moreadditional substitutions at one or more (e.g., several) other positions.

The amino acid changes may be of a minor nature, that is conservativeamino acid substitutions or insertions that do not significantly affectthe folding and/or activity of the protein; small deletions, typicallyof 1-30 amino acids; small amino- or carboxyl-terminal extensions, suchas an amino-terminal methionine residue; a small linker peptide of up to20-25 residues; or a small extension that facilitates purification bychanging net charge or another function, such as a poly-histidine tract,an antigenic epitope or a binding domain.

Examples of conservative substitutions are within the groups of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions that do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. Commonsubstitutions are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr,Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile,Leu/Val, Ala/Glu, and Asp/Gly.

Alternatively, the amino acid changes are of such a nature that thephysico-chemical properties of the polypeptides are altered. Forexample, amino acid changes may improve the thermal stability of thepolypeptide, alter the substrate specificity, change the pH optimum, andthe like.

Essential amino acids in a polypeptide can be identified according toprocedures known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, single alanine mutations areintroduced at every residue in the molecule, and the resultant mutantmolecules are tested for lipase activity to identify amino acid residuesthat are critical to the activity of the molecule. See also, Hilton etal., 1996, J. Biol. Chem. 271: 4699-4708. The active site of the enzymeor other biological interaction can also be determined by physicalanalysis of structure, as determined by such techniques as nuclearmagnetic resonance, crystallography, electron diffraction, orphotoaffinity labeling, in conjunction with mutation of putative contactsite amino acids. See, for example, de Vos et al., 1992, Science 255:306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver etal., 1992, FEBS Lett. 309: 59-64. The identity of essential amino acidscan also be inferred from an alignment with a related polypeptide.

The variants may consist or contain at least 50%, at least 55%, at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, or at least 95% of the number of amino acids of SEQID NO: 2.

Parent Lipases

The parent lipase may be selected from the group consisting of:

a) a polypeptide having at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99% or100% sequence identity to SEQ ID NO: 2;

b) a polypeptide encoded by a polynucleotide that hybridizes under lowstringency conditions, medium stringency conditions, medium-highstringency conditions, high stringency conditions, or very highstringency conditions with (i) the polypeptide coding sequence of SEQ IDNO: 1 or (ii) the full-length complement of (i);

c) a polypeptide encoded by a polynucleotide having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% sequence identity to SEQ ID NO: 1; and

d) a fragment of the polypeptide of SEQ ID NO: 2.

In an aspect of the invention, the parent lipase has a sequence identityto the polypeptide of SEQ ID NO: 2 of at least 60%, e.g., at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100%, which havelipase activity.

In one aspect, the amino acid sequence of the parent differs by up to 40amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, or 40 from the polypeptide of SEQ ID NO: 2.

In another aspect, the parent comprises or consists of the amino acidsequence of SEQ ID NO: 2.

In another aspect, the parent is a fragment of the polypeptide of SEQ IDNO: 2 containing at least 50%, at least 55%, at least 60%, at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, orat least 95% of the number of amino acids of SEQ ID NO: 2.

In another embodiment, the parent is an allelic variant of thepolypeptide of SEQ ID NO: 2.

In another aspect, the parent lipase is encoded by a polynucleotide thathybridizes under very low stringency conditions, low stringencyconditions, medium stringency conditions, medium-high stringencyconditions, high stringency conditions, or very high stringencyconditions with (i) the polypeptide coding sequence of SEQ ID NO: 1,(ii) the full-length complement of (i) (Sambrook et al., 1989, MolecularCloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, N.Y.).

The polynucleotide of SEQ ID NO: 1 or a subsequence thereof, as well asthe polypeptide of SEQ ID NO: 2 or a fragment thereof, may be used todesign nucleic acid probes to identify and clone DNA encoding a parentfrom strains of different genera or species according to methods wellknown in the art. In particular, such probes can be used forhybridization with the genomic DNA or cDNA of a cell of interest,following standard Southern blotting procedures, in order to identifyand isolate the corresponding gene therein. Such probes can beconsiderably shorter than the entire sequence, but should be at least15, e.g., at least 25, at least 35, or at least 70 nucleotides inlength. Preferably, the nucleic acid probe is at least 100 nucleotidesin length, e.g., at least 200 nucleotides, at least 300 nucleotides, atleast 400 nucleotides, at least 500 nucleotides, at least 600nucleotides, at least 700 nucleotides, at least 800 nucleotides, or atleast 900 nucleotides in length. Both DNA and RNA probes can be used.The probes are typically labeled for detecting the corresponding gene(for example, with ³²P, ³H, ³⁵S, biotin, or avidin). Such probes areencompassed by the present invention.

A genomic DNA or cDNA library prepared from such other strains may bescreened for DNA that hybridizes with the probes described above andencodes a parent. Genomic or other DNA from such other strains may beseparated by agarose or polyacrylamide gel electrophoresis, or otherseparation techniques. DNA from the libraries or the separated DNA maybe transferred to and immobilized on nitrocellulose or other suitablecarrier material. In order to identify a clone or DNA that hybridizeswith SEQ ID NO: 1 or a subsequence thereof, the carrier material is usedin a Southern blot.

For purposes of the present invention, hybridization indicates that thepolynucleotide hybridizes to a labeled nucleic acid probe correspondingto (i) SEQ ID NO: 1; (ii) the polypeptide coding sequence of SEQ ID NO:1; (iii) the full-length complement thereof; or (iv) a subsequencethereof; under very low to very high stringency conditions. Molecules towhich the nucleic acid probe hybridizes under these conditions can bedetected using, for example, X-ray film or any other detection meansknown in the art.

In one aspect, the nucleic acid probe is the polypeptide coding sequenceof SEQ ID NO: 1. In another aspect, the nucleic acid probe is at least50%, at least 55%, at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, or at least 95% of thenumber of nucleotides of SEQ ID NO: 1. In another aspect, the nucleicacid probe is a polynucleotide that encodes the polypeptide of SEQ IDNO: 2; the polypeptide thereof; or a fragment thereof. In anotheraspect, the nucleic acid probe is SEQ ID NO: 1.

In another embodiment, the parent is encoded by a polynucleotide havinga sequence identity to the polypeptide coding sequence of SEQ ID NO: 1of at least 60%, e.g., at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%.

The polypeptide may be a hybrid polypeptide in which a region of onepolypeptide is fused at the N-terminus or the C-terminus of a region ofanother polypeptide.

The parent lipase may be a fusion polypeptide or cleavable fusionpolypeptide in which another polypeptide is fused at the N-terminus orthe C-terminus of the polypeptide of the present invention. A fusionpolypeptide is produced by fusing a polynucleotide encoding anotherpolypeptide to a polynucleotide of the present invention. Techniques forproducing fusion polypeptides are known in the art, and include ligatingthe coding sequences encoding the polypeptides so that they are in frameand that expression of the fusion polypeptide is under control of thesame promoter(s) and terminator. Fusion polypeptides may also beconstructed using intein technology in which fusion polypeptides arecreated post-translationally (Cooper et al., 1993, EMBO J. 12:2575-2583; Dawson et al., 1994, Science 266: 776-779).

A fusion polypeptide can further comprise a cleavage site between thetwo polypeptides. Upon secretion of the fusion protein, the site iscleaved releasing the two polypeptides. Examples of cleavage sitesinclude, but are not limited to, the sites disclosed in Martin et al.,2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000,J. Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1997, Appl.Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13:498-503; and Contreras et al., 1991, Biotechnology 9: 378-381; Eaton etal., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995,Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure,Function, and Genetics 6: 240-248; and Stevens, 2003, Drug DiscoveryWorld 4: 35-48.

The parent lipase may be obtained from microorganisms of any genus. Forpurposes of the present invention, the term “obtained from” as usedherein in connection with a given source shall mean that the parentencoded by a polynucleotide is produced by the source or by a strain inwhich the polynucleotide from the source has been inserted. In oneaspect, the parent is secreted extracellularly.

The parent may be a bacterial lipase. For example, the parent may be aGram-positive bacterial polypeptide such as a Bacillus, Clostridium,Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus,Staphylococcus, Streptococcus, Streptomyces or Thermobifida lipase, or aGram-negative bacterial polypeptide such as a Campylobacter, E. coli,Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria,Pseudomonas, Salmonella, or Ureaplasma lipase.

In one aspect, the parent is a Bacillus alkalophilus, Bacillusamyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillusclausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacilluslentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus,Bacillus stearothermophilus, Bacillus subtilis, or Bacillusthuringiensis lipase.

In another aspect, the parent is a Streptococcus equisimilis,Streptococcus pyogenes, Streptococcus uberis, or Streptococcus equisubsp. Zooepidemicus lipase.

In another aspect, the parent is a Streptomyces achromogenes,Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus,or Streptomyces lividans lipase.

In another aspect, the parent is a Thermobifida alba or Thermobifidafusca (formerly known as Thermomonaspora fusca) lipase.

The parent may be a fungal lipase. For example, the parent may be ayeast lipase such as a Candida, Kluyveromyces, Pichia, Saccharomyces,Schizosaccharomyces, or Yarrowia lipase; or a filamentous fungal lipasesuch as an Acremonium, Agaricus, Alternaria, Aspergillus, Aureobasidium,Botryospaeria, Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps,Cochliobolus, Coprinopsis, Coptotermes, Corynascus, Cryphonectria,Cryptococcus, Diplodia, Exidia, Filibasidium, Fusarium, Gibberella,Holomastigotoides, Humicola, Irpex, Lentinula, Leptospaeria,Magnaporthe, Melanocarpus, Meripilus, Mucor, Myceliophthora,Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete,Piromyces, Poitrasia, Pseudoplectania, Pseudotrichonympha, Rhizomucor,Schizophyllum, Scytalidium, Talaromyces, Thermoascus, Thielavia,Tolypocladium, Trichoderma, Trichophaea, Verticillium, Volvariella, orXylaria lipase.

In another aspect, the parent is a Saccharomyces carlsbergensis,Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomycesdouglasfi, Saccharomyces kluyveri, Saccharomyces norbensis, orSaccharomyces oviformis lipase.

In another aspect, the parent is an Acremonium cellulolyticus,Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus,Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans,Aspergillus niger, Aspergillus oryzae, Chrysosporium inops,Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporiummerdarium, Chrysosporium pannicola, Chrysosporium queenslandicum,Chrysosporium tropicum, Chrysosporium zonaturn, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinurn, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa,Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurosporacrassa, Penicillium funiculosum, Penicillium purpurogenum, Phanerochaetechrysosporium, Thielavia achromatica, Thielavia albomyces, Thielaviaalbopilosa, Thielavia australeinsis, Thielavia fimeti, Thielaviamicrospora, Thielavia ovispora, Thielavia peruviana, Thielavia setosa,Thielavia spededonium, Thielavia subthermophila, Thielavia terrestris,Trichoderma harzianum, Trichoderma koningii, Trichodermalongibrachiatum, Trichoderma reesei, or Trichoderma viride lipase.

In another aspect, the parent is a Thermomyces lanuginosus lipase, e.g.,in particular the lipase of SEQ ID NO: 2.

It will be understood that for the aforementioned species, the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS),and Agricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

The parent lipase may be identified and obtained from other sourcesincluding microorganisms isolated from nature (e.g., soil, composts,water, etc.) or DNA samples obtained directly from natural materials(e.g., soil, composts, water, etc.) using the above-mentioned probes.Techniques for isolating microorganisms and DNA directly from naturalhabitats are well known in the art. A polynucleotide encoding a parentmay then be obtained by similarly screening a genomic DNA or cDNAlibrary of another microorganism or mixed DNA sample. Once apolynucleotide encoding a parent has been detected with the probe(s),the polynucleotide can be isolated or cloned by utilizing techniquesthat are known to those of ordinary skill in the art (see, e.g.,Sambrook et al., 1989, supra).

Preparation of Variants

The present invention also relates to methods for obtaining lipasevariants of the invention comprising: (a) introducing substitutions atpositions corresponding to G23S, D27N, A40I, F51I,L, E56R, D57N, K98I,R118F, G163S, T231R, N233R, Y220F, T244E, and P256T; (b) selecting thevariant which has lipase activity and in comparison with the parentlipase has one of the desired properties listed above; and (c)recovering the variant.

The variants can be prepared using any mutagenesis procedure known inthe art, such as site-directed mutagenesis, synthetic gene construction,semi-synthetic gene construction, random mutagenesis, shuffling, etc.

Site-directed mutagenesis is a technique in which one or more (e.g.,several) mutations are introduced at one or more defined sites in apolynucleotide encoding the parent lipase.

Site-directed mutagenesis can be accomplished in vitro by PCR involvingthe use of oligonucleotide primers containing the desired mutation.Site-directed mutagenesis can also be performed in vitro by cassettemutagenesis involving the cleavage by a restriction enzyme at a site inthe plasmid comprising a polynucleotide encoding the parent lipase andsubsequent ligation of an oligonucleotide containing the mutation in thepolynucleotide. Usually the restriction enzyme that digests the plasmidand the oligonucleotide is the same, permitting sticky ends of theplasmid and the insert to ligate to one another. See, e.g., Scherer andDavis, 1979, Proc. Natl. Acad. Sci. USA 76: 4949-4955; and Barton etal., 1990, Nucleic Acids Res. 18: 7349-4966.

Site-directed mutagenesis can also be accomplished in vivo by methodsknown in the art. See, e.g., US2004/0171154; Storici et al., 2001,Nature Biotechnol. 19: 773-776; Kren et al., 1998, Nat. Med. 4: 285-290;and Calissano and Macino, 1996, Fungal Genet. Newslett. 43: 15-16.

Any site-directed mutagenesis procedure can be used in the presentinvention. There are many commercial kits available that can be used toprepare variants.

Synthetic gene construction entails in vitro synthesis of a designedpolynucleotide molecule to encode a polypeptide of interest. Genesynthesis can be performed utilizing a number of techniques, such as themultiplex microchip-based technology described by Tian et al. (2004,Nature 432: 1050-1054) and similar technologies wherein oligonucleotidesare synthesized and assembled upon photo-programmable microfluidicchips.

Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO95/17413; or WO95/22625. Other methods that can be usedinclude error-prone PCR, phage display (e.g., Lowman et al., 1991,Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204) andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Neret al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide.

Semi-synthetic gene construction is accomplished by combining aspects ofsynthetic gene construction, and/or site-directed mutagenesis, and/orrandom mutagenesis, and/or shuffling. Semi-synthetic construction istypified by a process utilizing polynucleotide fragments that aresynthesized, in combination with PCR techniques. Defined regions ofgenes may thus be synthesized de novo, while other regions may beamplified using site-specific mutagenic primers, while yet other regionsmay be subjected to error-prone PCR or non-error prone PCRamplification. Polynucleotide subsequences may then be shuffled.

Polynucleotides

The present invention also relates to isolated polynucleotides encodinglipase variants of the present invention. In certain aspects, thepresent invention relates to a nucleic acid construct comprising thepolynucleotide of the invention. In certain aspects, the presentinvention relates to an expression vector comprising the polynucleotideof the invention. In certain aspects, the present invention relates to ahost cell comprising the polynucleotide of the invention. In certainaspects, the present invention relates to a method of producing a lipasevariant, comprising: (a) cultivating a host cell of the invention underconditions suitable for expression of the variant; and (b) recoveringthe variant.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisinga polynucleotide encoding a variant of the present invention operablylinked to one or more control sequences that direct the expression ofthe coding sequence in a suitable host cell under conditions compatiblewith the control sequences.

The polynucleotide may be manipulated in a variety of ways to providefor expression of a variant. Manipulation of the polynucleotide prior toits insertion into a vector may be desirable or necessary depending onthe expression vector. The techniques for modifying polynucleotidesutilizing recombinant DNA methods are well known in the art.

The control sequence may be a promoter, a polynucleotide which isrecognized by a host cell for expression of the polynucleotide. Thepromoter contains transcriptional control sequences that mediate theexpression of the variant. The promoter may be any polynucleotide thatshows transcriptional activity in the host cell including mutant,truncated, and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a bacterial hostcell are the promoters obtained from the Bacillus amyloliquefaciensalpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus licheniformis penicillinase gene (penP), Bacillusstearothermophilus maltogenic amylase gene (amyM), Bacillus subtilislevansucrase gene (sacB), Bacillus subtilis xylA and xylB genes,Bacillus thuringiensis cryIIIA gene (Agaisse and Lereclus, 1994,Molecular Microbiology 13: 97-107), E. coli lac operon, E. coli trcpromoter (Egon et al., 1988, Gene 69: 301-315), Streptomyces coelicoloragarase gene (dagA), and prokaryotic beta-lactamase gene (Villa-Kamaroffet al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731), as well as thetac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25). Further promoters are described in “Useful proteins fromrecombinant bacteria” in Gilbert et al., 1980, Scientific American 242:74-94; and in Sambrook et al., 1989, supra. Examples of tandem promotersare disclosed in WO 99/43835.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus nidulansacetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus nigeracid stable alpha-amylase, Aspergillus niger or Aspergillus awamoriglucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzaealkaline protease, Aspergillus oryzae triose phosphate isomerase,Fusarium oxysporum trypsin-like protease (WO96/00787), Fusariumvenenatum amyloglucosidase (WO00/56900), Fusarium venenatum Daria(WO00/56900), Fusarium venenatum Quinn (WO00/56900), Rhizomucor mieheilipase, Rhizomucor miehei aspartic proteinase, Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase IV, Trichoderma reeseiendoglucanase V, Trichoderma reesei xylanase I, Trichoderma reeseixylanase II, Trichoderma reesei beta-xylosidase, as well as the NA2-tpipromoter (a modified promoter from an Aspergillus neutral alpha-amylasegene in which the untranslated leader has been replaced by anuntranslated leader from an Aspergillus triose phosphate isomerase gene;non-limiting examples include modified promoters from an Aspergillusniger neutral alpha-amylase gene in which the untranslated leader hasbeen replaced by an untranslated leader from an Aspergillus nidulans orAspergillus oryzae triose phosphate isomerase gene); and mutant,truncated, and hybrid promoters thereof.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a transcription terminator, which isrecognized by a host cell to terminate transcription. The terminatorsequence is operably linked to the 3′-terminus of the polynucleotideencoding the variant. Any terminator that is functional in the host cellmay be used.

Preferred terminators for bacterial host cells are obtained from thegenes for Bacillus clausii alkaline protease (aprH), Bacilluslicheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA(rrnB).

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus nidulans anthranilate synthase,Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase,Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-likeprotease.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be an mRNA stabilizer region downstream ofa promoter and upstream of the coding sequence of a gene which increasesexpression of the gene.

Examples of suitable mRNA stabilizer regions are obtained from aBacillus thuringiensis cryIIIA gene (WO94/25612) and a Bacillus subtilisSP82 gene (Hue et al., 1995, Journal of Bacteriology 177: 3465-3471).

The control sequence may also be a leader, a nontranslated region of anmRNA that is important for translation by the host cell. The leadersequence is operably linked to the 5′-terminus of the polynucleotideencoding the variant. Any leader that is functional in the host cell maybe used.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′-terminus of the variant-encoding sequence and,when transcribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencethat is functional in the host cell may be used.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus nidulans anthranilatesynthase, Aspergillus niger glucoamylase, Aspergillus nigeralpha-glucosidase, Aspergillus oryzae TAKA amylase, and Fusariumoxysporum trypsin-like protease.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatencodes a signal peptide linked to the N-terminus of a variant anddirects the variant into the cell's secretory pathway. The 5′-end of thecoding sequence of the polynucleotide may inherently contain a signalpeptide coding sequence naturally linked in translation reading framewith the segment of the coding sequence that encodes the variant.Alternatively, the 5′-end of the coding sequence may contain a signalpeptide coding sequence that is foreign to the coding sequence. Aforeign signal peptide coding sequence may be required where the codingsequence does not naturally contain a signal peptide coding sequence.Alternatively, a foreign signal peptide coding sequence may simplyreplace the natural signal peptide coding sequence in order to enhancesecretion of the variant. However, any signal peptide coding sequencethat directs the expressed variant into the secretory pathway of a hostcell may be used.

Effective signal peptide coding sequences for bacterial host cells arethe signal peptide coding sequences obtained from the genes for BacillusNCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin,Bacillus licheniformis beta-lactamase, Bacillus stearothermophilusalpha-amylase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Effective signal peptide coding sequences for filamentous fungal hostcells are the signal peptide coding sequences obtained from the genesfor Aspergillus niger neutral amylase, Aspergillus niger glucoamylase,Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicolainsolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucormiehei aspartic proteinase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding sequences are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding sequence thatencodes a propeptide positioned at the N-terminus of a variant. Theresultant polypeptide is known as a proenzyme or propolypeptide (or azymogen in some cases). A propolypeptide is generally inactive and canbe converted to an active polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding sequence may be obtained from the genes for Bacillus subtilisalkaline protease (aprE), Bacillus subtilis neutral protease (nprT),Myceliophthora thermophila laccase (WO95/33836), Rhizomucor mieheiaspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

Where both signal peptide and propeptide sequences are present, thepropeptide sequence is positioned next to the N-terminus of the variantand the signal peptide sequence is positioned next to the N-terminus ofthe propeptide sequence.

It may also be desirable to add regulatory sequences that regulateexpression of the variant relative to the growth of the host cell.Examples of regulatory systems are those that cause expression of thegene to be turned on or off in response to a chemical or physicalstimulus, including the presence of a regulatory compound. Regulatorysystems in prokaryotic systems include the lac, tac, and trp operatorsystems. In yeast, the ADH2 system or GAL1 system may be used. Infilamentous fungi, the Aspergillus niger glucoamylase promoter,Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus oryzaeglucoamylase promoter may be used. Other examples of regulatorysequences are those that allow for gene amplification. In eukaryoticsystems, these regulatory sequences include the dihydrofolate reductasegene that is amplified in the presence of methotrexate, and themetallothionein genes that are amplified with heavy metals. In thesecases, the polynucleotide encoding the variant would be operably linkedwith the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a polynucleotide encoding a variant of the present invention,a promoter, and transcriptional and translational stop signals. Thevarious nucleotide and control sequences may be joined together toproduce a recombinant expression vector that may include one or moreconvenient restriction sites to allow for insertion or substitution ofthe polynucleotide encoding the variant at such sites. Alternatively,the polynucleotide may be expressed by inserting the polynucleotide or anucleic acid construct comprising the polynucleotide into an appropriatevector for expression. In creating the expression vector, the codingsequence is located in the vector so that the coding sequence isoperably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the polynucleotide. The choice of thevector will typically depend on the compatibility of the vector with thehost cell into which the vector is to be introduced. The vector may be alinear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell, it is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon, may be used.

The vector preferably contains one or more selectable markers thatpermit easy selection of transformed, transfected, transduced, or thelike cells. A selectable marker is a gene the product of which providesfor biocide or viral resistance, resistance to heavy metals, prototrophyto auxotrophs, and the like.

Examples of bacterial selectable markers are Bacillus licheniformis orBacillus subtilis dal genes, or markers that confer antibioticresistance such as ampicillin, chloramphenicol, kanamycin, neomycin,spectinomycin or tetracycline resistance. Suitable markers for yeasthost cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2,MET3, TRP1, and URA3. Selectable markers for use in a filamentous fungalhost cell include, but are not limited to, amdS (acetamidase), argB(ornithine carbamoyltransferase), bar (phosphinothricinacetyltransferase), hph (hygromycin phosphotransferase), niaD (nitratereductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfateadenyltransferase), and trpC (anthranilate synthase), as well asequivalents thereof. Preferred for use in an Aspergillus cell areAspergillus nidulans or Aspergillus oryzae amdS and pyrG genes and aStreptomyces hygroscopicus bar gene.

The vector preferably contains an element(s) that permits integration ofthe vector into the host cell's genome or autonomous replication of thevector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the variant or any other element ofthe vector for integration into the genome by homologous ornon-homologous recombination. Alternatively, the vector may containadditional polynucleotides for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should contain a sufficientnumber of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000base pairs, and 800 to 10,000 base pairs, which have a high degree ofsequence identity to the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell.

Furthermore, the integrational elements may be non-encoding or encodingpolynucleotides. On the other hand, the vector may be integrated intothe genome of the host cell by non-homologous recombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication that functions in a cell.The term “origin of replication” or “plasmid replicator” means apolynucleotide that enables a plasmid or vector to replicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAMß1 permittingreplication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANSI (Gems et al., 1991, Gene 98: 61-67; Cullen et al.,1987, Nucleic Acids Res. 15: 9163-9175; WO 00/24883). Isolation of theAMA1 gene and construction of plasmids or vectors comprising the genecan be accomplished according to the methods disclosed in WO 00/24883.

More than one copy of a polynucleotide of the present invention may beinserted into a host cell to increase production of a variant. Anincrease in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant host cells, comprisinga polynucleotide encoding a variant of the present invention operablylinked to one or more control sequences that direct the production of avariant of the present invention. A construct or vector comprising apolynucleotide is introduced into a host cell so that the construct orvector is maintained as a chromosomal integrant or as a self-replicatingextra-chromosomal vector as described earlier. The term “host cell”encompasses any progeny of a parent cell that is not identical to theparent cell due to mutations that occur during replication. The choiceof a host cell will to a large extent depend upon the gene encoding thevariant and its source.

The host cell may be any cell useful in the recombinant production of avariant, e.g., a prokaryote or a eukaryote.

The prokaryotic host cell may be any Gram-positive or Gram-negativebacterium. Gram-positive bacteria include, but are not limited to,Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus,Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, andStreptomyces. Gram-negative bacteria include, but are not limited to,Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter,Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

The bacterial host cell may be any Bacillus cell including, but notlimited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillusbrevis, Bacillus circulans, Bacillus clausfi, Bacillus coagulans,Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacilluslicheniformis, Bacillus megaterium, Bacillus pumilus, Bacillusstearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

The bacterial host cell may also be any Streptococcus cell including,but not limited to, Streptococcus equisimilis, Streptococcus pyogenes,Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

The bacterial host cell may also be any Streptomyces cell, including,but not limited to, Streptomyces achromogenes, Streptomyces avermitilis,Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividanscells.

The introduction of DNA into a Bacillus cell may be effected byprotoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen.Genet. 168: 111-115), competent cell transformation (see, e.g., Youngand Spizizen, 1961, J. Bacteriol. 81: 823-829, or Dubnau andDavidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), electroporation(see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), orconjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169:5271-5278). The introduction of DNA into an E. coli cell may be effectedby protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol.166: 557-580) or electroporation (see, e.g., Dower et al., 1988, NucleicAcids Res. 16: 6127-6145). The introduction of DNA into a Streptomycescell may be effected by protoplast transformation, electroporation (see,e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405),conjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171:3583-3585), or transduction (see, e.g., Burke et al., 2001, Proc. Natl.Acad. Sci. USA 98: 6289-6294). The introduction of DNA into aPseudomonas cell may be effected by electroporation (see, e.g., Choi etal., 2006, J. Microbiol. Methods 64: 391-397), or conjugation (see,e.g., Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71: 51-57). Theintroduction of DNA into a Streptococcus cell may be effected by naturalcompetence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32:1295-1297), protoplast transformation (see, e.g., Catt and Jollick,1991, Microbios 68: 189-207), electroporation (see, e.g., Buckley etal., 1999, Appl. Environ. Microbiol. 65: 3800-3804) or conjugation (see,e.g., Clewell, 1981, Microbiol. Rev. 45: 409-436). However, any methodknown in the art for introducing DNA into a host cell can be used.

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

The host cell may be a fungal cell. “Fungi” as used herein includes thephyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as wellas the Oomycota and all mitosporic fungi (as defined by Hawksworth etal., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition,1995, CAB International, University Press, Cambridge, UK).

The fungal host cell may be a yeast cell. “Yeast” as used hereinincludes ascosporogenous yeast (Endomycetales), basidiosporogenousyeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes).Since the classification of yeast may change in the future, for thepurposes of this invention, yeast shall be defined as described inBiology and Activities of Yeast (Skinner, Passmore, and Davenport,editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia,Saccharomyces, Schizosaccharomyces, or Yarrowia cell such as aKluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomycescerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii,Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomycesoviformis, or Yarrowia lipolytica cell.

The fungal host cell may be a filamentous fungal cell. “Filamentousfungi” include all filamentous forms of the subdivision Eumycota andOomycota (as defined by Hawksworth et al., 1995, supra). The filamentousfungi are generally characterized by a mycelial wall composed of chitin,cellulose, glucan, chitosan, mannan, and other complex polysaccharides.Vegetative growth is by hyphal elongation and carbon catabolism isobligately aerobic. In contrast, vegetative growth by yeasts such asSaccharomyces cerevisiae is by budding of a unicellular thallus andcarbon catabolism may be fermentative.

The filamentous fungal host cell may be an Acremonium, Aspergillus,Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus,Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe,Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trametes, or Trichoderma cell.

For example, the filamentous fungal host cell may be an Aspergillusawamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillusjaponicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea,Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsisrivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora,Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporiumlucknowense, Chrysosporium merdarium, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporiumzonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulaturn, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum,Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii,Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81:1470-1474, and Christensen et al., 1988, Bio/Technology 6: 1419-1422.Suitable methods for transforming Fusarium species are described byMalardier et al., 1989, Gene 78: 147-156, and WO96/00787. Yeast may betransformed using the procedures described by Becker and Guarente, InAbelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics andMolecular Biology, Methods in Enzymology, Volume 194, pp 182-187,Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153:163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.

Methods of Production

The present invention also relates to methods of producing a lipasevariant of the present invention, comprising: (a) cultivating a hostcell of the present invention under conditions suitable for expressionof the variant; and (b) recovering the variant.

The host cells are cultivated in a nutrient medium suitable forproduction of the variant using methods known in the art. For example,the cell may be cultivated by shake flask cultivation, or small-scale orlarge-scale fermentation (including continuous, batch, fed-batch, orsolid state fermentations) in laboratory or industrial fermentorsperformed in a suitable medium and under conditions allowing the variantto be expressed and/or isolated. The cultivation takes place in asuitable nutrient medium comprising carbon and nitrogen sources andinorganic salts, using procedures known in the art. Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). If the variant is secreted into the nutrient medium, thevariant can be recovered directly from the medium. If the variant is notsecreted, it can be recovered from cell lysates.

The variant may be detected using methods known in the art that arespecific for the variants. These detection methods include, but are notlimited to, use of specific antibodies, formation of an enzyme product,or disappearance of an enzyme substrate. For example, an enzyme assaymay be used to determine the activity of the variant such as thosedescribed in the examples.

The variant may be recovered using methods known in the art. Forexample, the variant may be recovered from the nutrient medium byconventional procedures including, but not limited to, collection,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation.

The variant may be purified by a variety of procedures known in the artincluding, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson andRyden, editors, VCH Publishers, New York, 1989) to obtain substantiallypure variants.

In an alternative aspect, the variant is not recovered, but rather ahost cell of the present invention expressing the variant is used as asource of the variant.

Compositions

The invention also includes compositions comprising the lipase variantof the present inventions.

In certain aspects the present invention relates to compositioncomprising a variant of a parent lipase which variant has lipaseactivity, has at least 60% but less than 100% sequence identity with SEQID NO: 2, and comprises one or more (e.g. several) substitutions atpositions corresponding to G23S, D27N, A40I, F51I,L, E56R, D57N, K98I,R118F, G163S, T231R, N233R, Y220F, T244E, and P256T.

In certain aspects the composition comprises substitutions at positionscorresponding to R118F+T231R+N233R and one or more (e.g., several)substitutions at positions corresponding to G23S, D27N, A40I, F51I,L,E56R, D57N, K98I, G163S, Y220F, T244E, and P256T.

In certain aspects said variant has improved wash performance, reducedodor-generation and/or improved storage stability/longer shelflife/increased thermostability.

The non-limiting list of composition components illustrated hereinafterare suitable for use in the compositions and methods herein may bedesirably incorporated in certain embodiments of the invention, e.g. toassist or enhance cleaning performance, for treatment of the substrateto be cleaned, or to modify the aesthetics of the composition as is thecase with perfumes, colorants, dyes or the like. The levels of any suchcomponents incorporated in any compositions are in addition to anymaterials previously recited for incorporation. The precise nature ofthese additional components, and levels of incorporation thereof, willdepend on the physical form of the composition and the nature of thecleaning operation for which it is to be used. Although componentsmentioned below are categorized by general header according to aparticular functionality, this is not to be construed as a limitation,as a component may comprise additional functionalities as will beappreciated by the skilled artisan.

Unless otherwise indicated the amounts in percentage is by weight of thecomposition (wt %). Suitable component materials include, but are notlimited to, surfactants, builders, chelating agents, dye transferinhibiting agents, dispersants, enzymes, and enzyme stabilizers,catalytic materials, bleach activators, hydrogen peroxide, sources ofhydrogen peroxide, preformed peracids, polymeric dispersing agents, claysoil removal/anti-redeposition agents, brighteners, suds suppressors,dyes, hueing dyes, perfumes, perfume delivery systems, structureelasticizing agents, fabric softeners, carriers, hydrotropes, processingaids, solvents and/or pigments. In addition to the disclosure below,suitable examples of such other components and levels of use are foundin U.S. Pat. Nos. 5,576,282, 6,306,812, and 6,326,348 herebyincorporated by reference.

Thus, in certain embodiments the invention do not contain one or more ofthe following adjuncts materials: surfactants, soaps, builders,chelating agents, dye transfer inhibiting agents, dispersants,additional enzymes, enzyme stabilizers, catalytic materials, bleachactivators, hydrogen peroxide, sources of hydrogen peroxide, preformedperacids, polymeric dispersing agents, clay soilremoval/anti-redeposition agents, brighteners, suds suppressors, dyes,perfumes, perfume delivery systems, structure elasticizing agents,fabric softeners, carriers, hydrotropes, processing aids, solventsand/or pigments. However, when one or more components are present, suchone or more components may be present as detailed below:

Surfactants—The compositions according to the present invention maycomprise a surfactant or surfactant system wherein the surfactant can beselected from nonionic surfactants, anionic surfactants, cationicsurfactants, ampholytic surfactants, zwitterionic surfactants,semi-polar nonionic surfactants and mixtures thereof. When present,surfactant is typically present at a level of from 0.1 to 60 wt %, from0.2 to 40 wt %, from 0.5 to 30 wt %, from 1 to 50 wt %, from 1 to 40 wt%, from 1 to 30 wt %, from 1 to 20 wt %, from 3 to 10 wt %, from 3 to 5wt %, from 5 to 40 wt %, from 5 to 30 wt %, from 5 to 15 wt %, from 3 to20 wt %, from 3 to 10 wt %, from 8 to 12 wt %, from 10 to 12 wt %, from20 to 25 wt % or from 25-60%.

Suitable anionic detersive surfactants include sulphate and sulphonatedetersive surfactants.

Suitable sulphonate detersive surfactants include alkyl benzenesulphonate, in one aspect, C₁₀₋₁₃ alkyl benzene sulphonate. Suitablealkyl benzene sulphonate (LAS) may be obtained, by sulphonatingcommercially available linear alkyl benzene (LAB); suitable LAB includeslow 2-phenyl LAB, such as Isochem® or Petrelab®, other suitable LABinclude high 2-phenyl LAB, such as Hyblene®. A suitable anionicdetersive surfactant is alkyl benzene sulphonate that is obtained byDETAL catalyzed process, although other synthesis routes, such as HF,may also be suitable. In one aspect a magnesium salt of LAS is used.

Suitable sulphate detersive surfactants include alkyl sulphate, in oneaspect, 0818 alkyl sulphate, or predominantly 012 alkyl sulphate.

Another suitable sulphate detersive surfactant is alkyl alkoxylatedsulphate, in one aspect, alkyl ethoxylated sulphate, in one aspect, aC₈₋₁₈ alkyl alkoxylated sulphate, in another aspect, a C₈₋₁₈ alkylethoxylated sulphate, typically the alkyl alkoxylated sulphate has anaverage degree of alkoxylation of from 0.5 to 20, or from 0.5 to 10,typically the alkyl alkoxylated sulphate is a C₈₋₁₈ alkyl ethoxylatedsulphate having an average degree of ethoxylation of from 0.5 to 10,from 0.5 to 7, from 0.5 to 5 or from 0.5 to 3.

The alkyl sulphate, alkyl alkoxylated sulphate and alkyl benzenesulphonates may be linear or branched, substituted or un-substituted.

The detersive surfactant may be a mid-chain branched detersivesurfactant, in one aspect, a mid-chain branched anionic detersivesurfactant, in one aspect, a mid-chain branched alkyl sulphate and/or amid-chain branched alkyl benzene sulphonate, e.g. a mid-chain branchedalkyl sulphate. In one aspect, the mid-chain branches are C₁₋₄ alkylgroups, typically methyl and/or ethyl groups.

Non-limiting examples of anionic surfactants include sulfates andsulfonates, in particular, linear alkylbenzenesulfonates (LAS), isomersof LAS, branched alkylbenzenesulfonates (BABS), phenylalkanesulfonates,alpha-olefinsulfonates (AOS), olefin sulfonates, alkene sulfonates,alkane-2,3-diylbis(sulfates), hydroxyalkanesulfonates and disulfonates,alkyl sulfates (AS) such as sodium dodecyl sulfate (SDS), fatty alcoholsulfates (FAS), primary alcohol sulfates (PAS), alcohol ethersulfates(AES or AEOS or FES, also known as alcohol ethoxysulfates or fattyalcohol ether sulfates), secondary alkanesulfonates (SAS), paraffinsulfonates (PS), ester sulfonates, sulfonated fatty acid glycerolesters, alpha-sulfo fatty acid methyl esters (alpha-SFMe or SES)including methyl ester sulfonate (MES), alkyl- or alkenylsuccinic acid,dodecenyl/tetradecenyl succinic acid (DTSA), fatty acid derivatives ofamino acids, diesters and monoesters of sulfo-succinic acid or soap, andcombinations thereof.

Suitable non-ionic detersive surfactants are selected from the groupconsisting of: C₈-C₁₈ alkyl ethoxylates, such as, NEODOL®; C₆-C₁₂ alkylphenol alkoxylates wherein the alkoxylate units may be ethyleneoxyunits, propyleneoxy units or a mixture thereof; C₁₂-C₁₈ alcohol andC₆-C₁₂ alkyl phenol condensates with ethylene oxide/propylene oxideblock polymers such as Pluronic®; C₁₄-C₂₂ mid-chain branched alcohols;C₁₄-C₂₂ mid-chain branched alkyl alkoxylates, typically having anaverage degree of alkoxylation of from 1 to 30; alkylpolysaccharides, inone aspect, alkylpolyglycosides; polyhydroxy fatty acid amides; ethercapped poly(oxyalkylated) alcohol surfactants; and mixtures thereof.

Suitable non-ionic detersive surfactants include alkyl polyglucosideand/or an alkyl alkoxylated alcohol.

In one aspect, non-ionic detersive surfactants include alkyl alkoxylatedalcohols, in one aspect C₈₋₁₈ alkyl alkoxylated alcohol, e.g. a C₈₋₁₈alkyl ethoxylated alcohol, the alkyl alkoxylated alcohol may have anaverage degree of alkoxylation of from 1 to 50, from 1 to 30, from 1 to20, or from 1 to 10. In one aspect, the alkyl alkoxylated alcohol may bea C₈₋₁₈ alkyl ethoxylated alcohol having an average degree ofethoxylation of from 1 to 10, from 1 to 7, more from 1 to 5 or from 3 to7. The alkyl alkoxylated alcohol can be linear or branched, andsubstituted or un-substituted. Suitable nonionic surfactants includeLutensol®.

Non-limiting examples of nonionic surfactants include alcoholethoxylates (AE or AEO), alcohol propoxylates, propoxylated fattyalcohols (PFA), alkoxylated fatty acid alkyl esters, such as ethoxylatedand/or propoxylated fatty acid alkyl esters, alkylphenol ethoxylates(APE), nonylphenol ethoxylates (NPE), alkylpolyglycosides (APG),alkoxylated amines, fatty acid monoethanolamides (FAM), fatty aciddiethanolamides (FADA), ethoxylated fatty acid monoethanolamides (EFAM),propoxylated fatty acid monoethanolamides (PFAM), polyhydroxyalkyl fattyacid amides, or N-acyl N-alkyl derivatives of glucosamine (glucamides,GA, or fatty acid glucamides, FAGA), as well as products available underthe trade names SPAN and TWEEN, and combinations thereof.

Suitable cationic detersive surfactants include alkyl pyridiniumcompounds, alkyl quaternary ammonium compounds, alkyl quaternaryphosphonium compounds, alkyl ternary sulphonium compounds, and mixturesthereof.

Suitable cationic detersive surfactants are quaternary ammoniumcompounds having the general formula: (R)(R₁)(R₂)(R₃)N⁺X⁻, wherein, R isa linear or branched, substituted or unsubstituted C₆₋₁₈ alkyl oralkenyl moiety, R₁ and R₂ are independently selected from methyl orethyl moieties, R₃ is a hydroxyl, hydroxymethyl or a hydroxyethylmoiety, X is an anion which provides charge neutrality, suitable anionsinclude: halides, e.g. chloride; sulphate; and sulphonate. Suitablecationic detersive surfactants are mono-C₈₋₁₈ alkyl mono-hydroxyethyldi-methyl quaternary ammonium chlorides. Highly suitable cationicdetersive surfactants are mono-C₈₋₁₀ alkyl mono-hydroxyethyl di-methylquaternary ammonium chloride, mono-C₁₀₋₁₂ alkyl mono-hydroxyethyldi-methyl quaternary ammonium chloride and mono-C₁₀ alkylmono-hydroxyethyl di-methyl quaternary ammonium chloride.

Non-limiting examples of cationic surfactants includealkyldimethylethanolamine quat (ADMEAQ), cetyltrimethylammonium bromide(CTAB), dimethyldistearylammonium chloride (DSDMAC), andalkylbenzyldimethylammonium, alkyl quaternary ammonium compounds,alkoxylated quaternary ammonium (AQA) compounds, ester quats, andcombinations thereof.

Suitable amphoteric/zwitterionic surfactants include amine oxides andbetaines such as alkyldimethylbetaines, sulfobetaines, or combinationsthereof. Amine-neutralized anionic surfactants—Anionic surfactants ofthe present invention and adjunct anionic cosurfactants, may exist in anacid form, and said acid form may be neutralized to form a surfactantsalt which is desirable for use in the present detergent compositions.Typical agents for neutralization include the metal counterion base suchas hydroxides, eg, NaOH or KOH. Further preferred agents forneutralizing anionic surfactants of the present invention and adjunctanionic surfactants or cosurfactants in their acid forms includeammonia, amines, or alkanolamines. Alkanolamines are preferred. Suitablenon-limiting examples including monoethanolamine, diethanolamine,triethanolamine, and other linear or branched alkanolamines known in theart; e.g., highly preferred alkanolamines include 2-amino-1-propanol,1-aminopropanol, monoisopropanolamine, or 1-amino-3-propanol. Amineneutralization may be done to a full or partial extent, e.g. part of theanionic surfactant mix may be neutralized with sodium or potassium andpart of the anionic surfactant mix may be neutralized with amines oralkanolamines.

Non-limiting examples of semipolar surfactants include amine oxides (AO)such as alkyldimethylamineoxide

Surfactant systems comprising mixtures of one or more anionic and inaddition one or more nonionic surfactants optionally with an additionalsurfactant such as a cationic surfactant, may be preferred. Preferredweight ratios of anionic to nonionic surfactant are at least 2:1, or atleast 1:1 to 1:10.

In one aspect a surfactant system may comprise a mixture of isoprenoidsurfactants represented by formula A and formula B:

where Y is CH₂ or null, and Z may be chosen such that the resultingsurfactant is selected from the following surfactants: an alkylcarboxylate surfactant, an alkyl polyalkoxy surfactant, an alkyl anionicpolyalkoxy sulfate surfactant, an alkyl glycerol ester sulfonatesurfactant, an alkyl dimethyl amine oxide surfactant, an alkylpolyhydroxy based surfactant, an alkyl phosphate ester surfactant, analkyl glycerol sulfonate surfactant, an alkyl polygluconate surfactant,an alkyl polyphosphate ester surfactant, an alkyl phosphonatesurfactant, an alkyl polyglycoside surfactant, an alkyl monoglycosidesurfactant, an alkyl diglycoside surfactant, an alkyl sulfosuccinatesurfactant, an alkyl disulfate surfactant, an alkyl disulfonatesurfactant, an alkyl sulfosuccinamate surfactant, an alkyl glucamidesurfactant, an alkyl taurinate surfactant, an alkyl sarcosinatesurfactant, an alkyl glycinate surfactant, an alkyl isethionatesurfactant, an alkyl dialkanolamide surfactant, an alkylmonoalkanolamide surfactant, an alkyl monoalkanolamide sulfatesurfactant, an alkyl diglycolamide surfactant, an alkyl diglycolamidesulfate surfactant, an alkyl glycerol ester surfactant, an alkylglycerol ester sulfate surfactant, an alkyl glycerol ether surfactant,an alkyl glycerol ether sulfate surfactant, alkyl methyl ester sulfonatesurfactant, an alkyl polyglycerol ether surfactant, an alkylpolyglycerol ether sulfate surfactant, an alkyl sorbitan estersurfactant, an alkyl ammonioalkanesulfonate surfactant, an alkylamidopropyl betaine surfactant, an alkyl allylated quat basedsurfactant, an alkyl monohydroxyalkyl-di-alkylated quat basedsurfactant, an alkyl di-hydroxyalkyl monoalkyl quat based surfactant, analkylated quat surfactant, an alkyl trimethylammonium quat surfactant,an alkyl polyhydroxalkyl oxypropyl quat based surfactant, an alkylglycerol ester quat surfactant, an alkyl glycol amine quat surfactant,an alkyl monomethyl dihydroxyethyl quaternary ammonium surfactant, analkyl dimethyl monohydroxyethyl quaternary ammonium surfactant, an alkyltrimethylammonium surfactant, an alkyl imidazoline-based surfactant, analken-2-yl-succinate surfactant, an alkyl a-sulfonated carboxylic acidsurfactant, an alkyl a-sulfonated carboxylic acid alkyl estersurfactant, an alpha olefin sulfonate surfactant, an alkyl phenolethoxylate surfactant, an alkyl benzenesulfonate surfactant, an alkylsulfobetaine surfactant, an alkyl hydroxysulfobetaine surfactant, analkyl ammoniocarboxylate betaine surfactant, an alkyl sucrose estersurfactant, an alkyl alkanolamide surfactant, an alkyldi(polyoxyethylene) monoalkyl ammonium surfactant, an alkylmono(polyoxyethylene) dialkyl ammonium surfactant, an alkyl benzyldimethylammonium surfactant, an alkyl aminopropionate surfactant, analkyl amidopropyl dimethylamine surfactant, or a mixture thereof; and ifZ is a charged moiety, Z is charge-balanced by a suitable metal ororganic counter ion. Suitable counter ions include a metal counter ion,an amine, or an alkanolamine, e.g., C1-C6 alkanolammonium. Morespecifically, suitable counter ions include Na+, Ca+, Li+, K+, Mg+,e.g., monoethanolamine (MEA), diethanolamine (DEA), triethanolamine(TEA), 2-amino-1-propanol, 1-aminopropanol, methyldiethanolamine,dimethylethanolamine, monoisopropanolamine, triisopropanolamine,1-amino-3-propanol, or mixtures thereof. In one embodiment, thecompositions contain from 5% to 97% of one or more non-isoprenoidsurfactants; and one or more adjunct cleaning additives; wherein theweight ratio of surfactant of formula A to surfactant of formula B isfrom 50:50 to 95:5.

Soap—The compositions herein may contain soap. Without being limited bytheory, it may be desirable to include soap as it acts in part as asurfactant and in part as a builder and may be useful for suppression offoam and may furthermore interact favorably with the various cationiccompounds of the composition to enhance softness on textile fabricstreaded with the inventive compositions. Any soap known in the art foruse in laundry detergents may be utilized. In one embodiment, thecompositions contain from 0 wt % to 20 wt %, from 0.5 wt % to 20 wt %,from 4 wt % to 10 wt %, or from 4 wt % to 7 wt % of soap.

Examples of soap useful herein include oleic acid soaps, palmitic acidsoaps, palm kernel fatty acid soaps, and mixtures thereof. Typical soapsare in the form of mixtures of fatty acid soaps having different chainlengths and degrees of substitution. One such mixture is topped palmkernel fatty acid.

In one embodiment, the soap is selected from free fatty acid. Suitablefatty acids are saturated and/or unsaturated and can be obtained fromnatural sources such a plant or animal esters (e.g., palm kernel oil,palm oil, coconut oil, babassu oil, safflower oil, tall oil, castor oil,tallow and fish oils, grease, and mixtures thereof), or syntheticallyprepared (e.g., via the oxidation of petroleum or by hydrogenation ofcarbon monoxide via the Fisher Tropsch process).

Examples of suitable saturated fatty acids for use in the compositionsof this invention include captic, lauric, myristic, palmitic, stearic,arachidic and behenic acid. Suitable unsaturated fatty acid speciesinclude: palmitoleic, oleic, linoleic, linolenic and ricinoleic acid.Examples of preferred fatty acids are saturated Cn fatty acid, saturatedCi₂-Ci₄ fatty acids, and saturated or unsaturated Cn to Ci₈ fatty acids,and mixtures thereof.

When present, the weight ratio of fabric softening cationic cosurfactantto fatty acid is preferably from about 1:3 to about 3:1, more preferablyfrom about 1:1.5 to about 1.5:1, most preferably about 1:1.

Levels of soap and of nonsoap anionic surfactants herein are percentagesby weight of the detergent composition, specified on an acid form basis.However, as is commonly understood in the art, anionic surfactants andsoaps are in practice neutralized using sodium, potassium oralkanolammonium bases, such as sodium hydroxide or monoethanolamine.

Hydrotropes—The compositions of the present invention may comprise oneor more hydrotropes. A hydrotrope is a compound that solubiliseshydrophobic compounds in aqueous solutions (or oppositely, polarsubstances in a non-polar environment). Typically, hydrotropes have bothhydrophilic and a hydrophobic character (so-called amphiphilicproperties as known from surfactants); however the molecular structureof hydrotropes generally do not favor spontaneous self-aggregation, seee.g. review by Hodgdon and Kaler (2007), Current Opinion in Colloid &Interface Science 12: 121-128. Hydrotropes do not display a criticalconcentration above which self-aggregation occurs as found forsurfactants and lipids forming miceller, lamellar or other well definedmeso-phases. Instead, many hydrotropes show a continuous-typeaggregation process where the sizes of aggregates grow as concentrationincreases. However, many hydrotropes alter the phase behavior,stability, and colloidal properties of systems containing substances ofpolar and non-polar character, including mixtures of water, oil,surfactants, and polymers. Hydrotropes are classically used acrossindustries from pharma, personal care, food, to technical applications.Use of hydrotropes in detergent compositions allow for example moreconcentrated formulations of surfactants (as in the process ofcompacting liquid detergents by removing water) without inducingundesired phenomena such as phase separation or high viscosity.

The detergent may contain from 0 to 10 wt %, such as from 0 to 5 wt %,0.5 to 5 wt %, or from 3% to 5 wt %, of a hydrotrope. Any hydrotropeknown in the art for use in detergents may be utilized. Non-limitingexamples of hydrotropes include sodium benzenesulfonate, sodiump-toluene sulfonate (STS), sodium xylene sulfonate (SXS), sodium cumenesulfonate (SCS), sodium cymene sulfonate, amine oxides, alcohols andpolyglycolethers, sodium hydroxynaphthoate, sodium hydroxynaphthalenesulfonate, sodium ethylhexyl sulfate, and combinations thereof.

Builders—The compositions of the present invention may comprise one ormore builders, co-builders, builder systems ora mixture thereof. When abuilder is used, the cleaning composition will typically comprise from 0to 65 wt %, at least 1 wt %, from 2 to 60 wt % or from 5 to 10 wt %builder. In a dish wash cleaning composition, the level of builder istypically 40 to 65 wt % or 50 to 65 wt %. The composition may besubstantially free of builder; substantially free means “no deliberatelyadded” zeolite and/or phosphate. Typical zeolite builders includezeolite A, zeolite P and zeolite MAP. A typical phosphate builder issodium tri-polyphosphate.

The builder and/or co-builder may particularly be a chelating agent thatforms water-soluble complexes with Ca and Mg. Any builder and/orco-builder known in the art for use in detergents may be utilized.Non-limiting examples of builders include zeolites, diphosphates(pyrophosphates), triphosphates such as sodium triphosphate (STP orSTPP), carbonates such as sodium carbonate, soluble silicates such assodium metasilicate, layered silicates (e.g., SKS-6 from Hoechst),ethanolamines such as 2-aminoethan-1-ol (MEA), iminodiethanol (DEA) and2,2′,2″-nitrilotriethanol (TEA), and carboxymethylinulin (CMI), andcombinations thereof.

The cleaning composition may include a co-builder alone, or incombination with a builder, e.g. a zeolite builder. Non-limitingexamples of co-builders include homopolymers of polyacrylates orcopolymers thereof, such as poly(acrylic acid) (PAA) or copoly(acrylicacid/maleic acid) (PAA/PMA). Further non-limiting examples includecitrate, chelators such as aminocarboxylates, aminopolycarboxylates andphosphonates, and alkyl- or alkenylsuccinic acid. Additional specificexamples include 2,2′,2″-nitrilotriacetic acid (NTA),etheylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaaceticacid (DTPA), iminodisuccinic acid (IDS), ethylenediamine-N,N′-disuccinicacid (EDDS), methylglycinediacetic acid (MGDA), glutamicacid-N,N-diacetic acid (GLDA), 1-hydroxyethane-1,1-diylbis(phosphonicacid) (HEDP), ethylenediaminetetrakis(methylene)tetrakis(phosphonicacid) (EDTMPA), diethylenetriaminepentakis(methylene)pentakis(phosphonicacid) (DTPMPA), N-(2-hydroxyethyl)iminodiacetic acid (EDG), asparticacid-N-monoacetic acid (ASMA), aspartic acid-N,N-diacetic acid (ASDA),aspartic acid-N-monopropionic acid (ASMP), iminodisuccinic acid (IDA),N-(2-sulfomethyl) aspartic acid (SMAS), N-(2-sulfoethyl) aspartic acid(SEAS), N-(2-sulfomethyl) glutamic acid (SMGL), N-(2-sulfoethyl)glutamic acid (SEGL), N-methyliminodiacetic acid (MIDA),α-alanine-N,N-diacetic acid (α-ALDA), serine-N,N-diacetic acid (SEDA),isoserine-N,N-diacetic acid (ISDA), phenylalanine-N,N-diacetic acid(PHDA), anthranilic acid-N,N-diacetic acid (ANDA), sulfanilic acid-N,N-diacetic acid (SLDA), taurine-N, N-diacetic acid (TUDA) andsulfomethyl-N,N-diacetic acid (SMDA),N-(hydroxyethyl)-ethylidenediaminetriacetate (HEDTA), diethanolglycine(DEG), Diethylenetriamine Penta (Methylene Phosphonic acid) (DTPMP),aminotris(methylenephosphonic acid) (ATMP), and combinations and saltsthereof. Further exemplary builders and/or co-builders are described in,e.g., WO09/102854, U.S. Pat. No. 5,977,053.

Chelating Agents and Crystal Growth Inhibitors—The compositions hereinmay contain a chelating agent and/or a crystal growth inhibitor.Suitable molecules include copper, iron and/or manganese chelatingagents and mixtures thereof. Suitable molecules include DTPA (Diethylenetriamine pentaacetic acid), HEDP (Hydroxyethane diphosphonic acid),DTPMP (Diethylene triamine penta(methylene phosphonic acid)),1,2-Dihydroxybenzene-3,5-disulfonic acid disodium salt hydrate,ethylenediamine, diethylene triamine, ethylenediaminedisuccinic acid(EDDS), N-hydroxyethylethylenediaminetri-acetic acid (HEDTA),triethylenetetraaminehexaacetic acid (TTHA), N-hydroxyethyliminodiaceticacid (HEIDA), dihydroxyethylglycine (DHEG),ethylenediaminetetrapropionic acid (EDTP), carboxymethyl inulin and2-Phosphonobutane 1,2,4-tricarboxylic acid (Bayhibit® AM) andderivatives thereof. Typically the composition may comprise from 0.005to 15 wt % or from 3.0 to 10 wt % chelating agent or crystal growthinhibitor.

Bleach Component—The bleach component suitable for incorporation in themethods and compositions of the invention comprise one or a mixture ofmore than one bleach component. Suitable bleach components includebleaching catalysts, photobleaches, bleach activators, hydrogenperoxide, sources of hydrogen peroxide, pre-formed peracids and mixturesthereof. In general, when a bleach component is used, the compositionsof the present invention may comprise from 0 to 30 wt %, from 0.00001 to90 wt %, 0.0001 to 50 wt %, from 0.001 to 25 wt % or from 1 to 20 wt %.Examples of suitable bleach components include:

(1) Pre-formed peracids: Suitable preformed peracids include, but arenot limited to, compounds selected from the group consisting ofpre-formed peroxyacids or salts thereof, typically either aperoxycarboxylic acid or salt thereof, or a peroxysulphonic acid or saltthereof.

The pre-formed peroxyacid or salt thereof is preferably aperoxycarboxylic acid or salt thereof, typically having a chemicalstructure corresponding to the following chemical formula:

wherein: R¹⁴ is selected from alkyl, aralkyl, cycloalkyl, aryl orheterocyclic groups; the R¹⁴ group can be linear or branched,substituted or unsubstituted; and Y is any suitable counter-ion thatachieves electric charge neutrality, preferably Y is selected fromhydrogen, sodium or potassium. Preferably, R¹⁴ is a linear or branched,substituted or unsubstituted C₆₋₉ alkyl. Preferably, the peroxyacid orsalt thereof is selected from peroxyhexanoic acid, peroxyheptanoic acid,peroxyoctanoic acid, peroxynonanoic acid, peroxydecanoic acid, any saltthereof, or any combination thereof. Particularly preferred peroxyacidsare phthalimido-peroxy-alkanoic acids, in particular ε-phthahlimidoperoxy hexanoic acid (PAP). Preferably, the peroxyacid or salt thereofhas a melting point in the range of from 30° C. to 60° C.

The pre-formed peroxyacid or salt thereof can also be a peroxysulphonicacid or salt thereof, typically having a chemical structurecorresponding to the following chemical formula:

wherein: R¹⁵ is selected from alkyl, aralkyl, cycloalkyl, aryl orheterocyclic groups; the R¹⁵ group can be linear or branched,substituted or unsubstituted; and Z is any suitable counter-ion thatachieves electric charge neutrality, preferably Z is selected fromhydrogen, sodium or potassium. Preferably R¹⁵ is a linear or branched,substituted or unsubstituted C₆₋₉ alkyl. Preferably such bleachcomponents may be present in the compositions of the invention in anamount from 0.01 to 50 wt % or from 0.1 to 20 wt %.

(2) Sources of hydrogen peroxide include e.g., inorganic perhydratesalts, including alkali metal salts such as sodium salts of perborate(usually mono- or tetra-hydrate), percarbonate, persulphate,perphosphate, persilicate salts and mixtures thereof. In one aspect ofthe invention the inorganic perhydrate salts such as those selected fromthe group consisting of sodium salts of perborate, percarbonate andmixtures thereof. When employed, inorganic perhydrate salts aretypically present in amounts of 0.05 to 40 wt % or 1 to 30 wt % of theoverall composition and are typically incorporated into suchcompositions as a crystalline solid that may be coated. Suitablecoatings include: inorganic salts such as alkali metal silicate,carbonate or borate salts or mixtures thereof, or organic materials suchas water-soluble or dispersible polymers, waxes, oils or fatty soaps.Preferably such bleach components may be present in the compositions ofthe invention in an amount of 0.01 to 50 wt % or 0.1 to 20 wt %.

(3) The term bleach activator is meant herein as a compound which reactswith hydrogen peroxide to form a peracid via perhydrolysis. The peracidthus formed constitutes the activated bleach. Suitable bleach activatorsto be used herein include those belonging to the class of esters,amides, imides or anhydrides. Suitable bleach activators are thosehaving R—(C═O)—L wherein R is an alkyl group, optionally branched,having, when the bleach activator is hydrophobic, from 6 to 14 carbonatoms, or from 8 to 12 carbon atoms and, when the bleach activator ishydrophilic, less than 6 carbon atoms or less than 4 carbon atoms; and Lis leaving group. Examples of suitable leaving groups are benzoic acidand derivatives thereof—especially benzene sulphonate. Suitable bleachactivators include dodecanoyl oxybenzene sulphonate, decanoyl oxybenzenesulphonate, decanoyl oxybenzoic acid or salts thereof, 3,5,5-trimethylhexanoyloxybenzene sulphonate, tetraacetyl ethylene diamine (TAED),sodium 4-[(3,5,5-trimethylhexanoyl)oxy]benzene-1-sulfonate (ISONOBS),4-(dodecanoyloxy)benzene-1-sulfonate (LOBS),4-(decanoyloxy)benzene-1-sulfonate, 4-(decanoyloxy)benzoate (DOBS orDOBA), 4-(nonanoyloxy)benzene-1-sulfonate (NOBS), and/or those disclosedin WO98/17767. A family of bleach activators is disclosed in EP624154and particularly preferred in that family is acetyl triethyl citrate(ATC). ATC or a short chain triglyceride like triacetin has theadvantage that it is environmentally friendly. Furthermore acetyltriethyl citrate and triacetin have good hydrolytical stability in theproduct upon storage and are efficient bleach activators. Finally ATC ismultifunctional, as the citrate released in the perhydrolysis reactionmay function as a builder. Alternatively, the bleaching system maycomprise peroxyacids of, for example, the amide, imide, or sulfone type.The bleaching system may also comprise peracids such as6-(phthalimido)peroxyhexanoic acid (PAP). Suitable bleach activators arealso disclosed in WO98/17767. While any suitable bleach activator may beemployed, in one aspect of the invention the subject cleaningcomposition may comprise NOBS, TAED or mixtures thereof. When present,the peracid and/or bleach activator is generally present in thecomposition in an amount of 0.1 to 60 wt %, 0.5 to 40 wt % or 0.6 to 10wt % based on the fabric and home care composition. One or morehydrophobic peracids or precursors thereof may be used in combinationwith one or more hydrophilic peracid or precursor thereof. Preferablysuch bleach components may be present in the compositions of theinvention in an amount of 0.01 to 50 wt %, or 0.1 to 20 wt %.

The amounts of hydrogen peroxide source and peracid or bleach activatormay be selected such that the molar ratio of available oxygen (from theperoxide source) to peracid is from 1:1 to 35:1, or even 2:1 to 10:1.

(4) Diacyl peroxides—preferred diacyl peroxide bleaching species includethose selected from diacyl peroxides of the general formula:R¹—C(O)—OO—(O)C—R², in which R¹ represents a C₆-C₁₈ alkyl, preferablyC₆-C₁₂ alkyl group containing a linear chain of at least 5 carbon atomsand optionally containing one or more substituents (e.g. —N⁺(CH₃)₃,—COOH or —CN) and/or one or more interrupting moieties (e.g. —CONH— or—CH═CH—) interpolated between adjacent carbon atoms of the alkylradical, and R² represents an aliphatic group compatible with a peroxidemoiety, such that R¹ and R² together contain a total of 8 to 30 carbonatoms. In one preferred aspect R¹ and R² are linear unsubstituted C₆-C₁₂alkyl chains. Most preferably R¹ and R² are identical. Diacyl peroxides,in which both R¹ and R² are C₆-C₁₂ alkyl groups, are particularlypreferred. Preferably, at least one of, most preferably only one of, theR groups (R₁ or R₂), does not contain branching or pendant rings in thealpha position, or preferably neither in the alpha nor beta positions ormost preferably in none of the alpha or beta or gamma positions. In onefurther preferred embodiment the DAP may be asymmetric, such thatpreferably the hydrolysis of R1 acyl group is rapid to generate peracid,but the hydrolysis of R2 acyl group is slow.

The tetraacyl peroxide bleaching species is preferably selected fromtetraacyl peroxides of the general formula:R³—C(O)—OO—O(O)—(CH₂)n-C(O)—OO—O(O)—R³, in which R³ represents a C₁-C₉alkyl, or C₃-C₇, group and n represents an integer from 2 to 12, or 4 to10 inclusive.

Preferably, the diacyl and/or tetraacyl peroxide bleaching species ispresent in an amount sufficient to provide at least 0.5 ppm, at least 10ppm, or at least 50 ppm by weight of the wash liquor. In a preferredembodiment, the bleaching species is present in an amount sufficient toprovide from 0.5 to 300 ppm, from 30 to 150 ppm by weight of the washliquor.

Preferably the bleach component comprises a bleach catalyst (5 and 6).

(5) Preferred are organic (non-metal) bleach catalysts include bleachcatalyst capable of accepting an oxygen atom from a peroxyacid and/orsalt thereof, and transferring the oxygen atom to an oxidizeablesubstrate. Suitable bleach catalysts include, but are not limited to:iminium cations and polyions; iminium zwitterions; modified amines;modified amine oxides; N-sulphonyl imines; N-phosphonyl imines; N-acylimines; thiadiazole dioxides; perfluoroimines; cyclic sugar ketones andmixtures thereof.

Suitable iminium cations and polyions include, but are not limited to,N-methyl-3,4-dihydroisoquinolinium tetrafluoroborate, prepared asdescribed in Tetrahedron (1992), 49(2), 423-38 (e.g. compound 4, p.433); N-methyl-3,4-dihydroisoquinolinium p-toluene sulphonate, preparedas described in U.S. Pat. No. 5,360,569 (e.g. Column 11, Example 1); andN-octyl-3,4-dihydroisoquinolinium p-toluene sulphonate, prepared asdescribed in U.S. Pat. No. 5,360,568 (e.g. Column 10, Ex. 3).

Suitable iminium zwitterions include, but are not limited to,N-(3-sulfopropyl)-3,4-dihydroisoquinolinium, inner salt, prepared asdescribed in U.S. Pat. No. 5,576,282 (e.g. Column 31, Ex. II);N-[2-(sulphooxy)dodecyl]-3,4-dihydroisoquinolinium, inner salt, preparedas described in U.S. Pat. No. 5,817,614 (e.g. Column 32, Ex. V);2-[3-[(2-ethylhexyl)oxy]-2-(sulphooxy)propyl]-3,4-dihydroisoquinolinium,inner salt, prepared as described in WO05/047264 (e.g. p. 18, Ex. 8),and2-[3-[(2-butyloctyl)oxy]-2-(sulphooxy)propyl]-3,4-dihydroisoquinolinium,inner salt.

Suitable modified amine oxygen transfer catalysts include, but are notlimited to, 1,2,3,4-tetrahydro-2-methyl-1-isoquinolinol, which can bemade according to the procedures described in Tetrahedron Letters(1987), 28(48), 6061-6064. Suitable modified amine oxide oxygen transfercatalysts include, but are not limited to, sodium1-hydroxy-N-oxy-N-[2-(sulphooxy)decyl]-1,2,3,4-tetrahydroisoquinoline.

Suitable N-sulphonyl imine oxygen transfer catalysts include, but arenot limited to, 3-methyl-1,2-benzisothiazole 1,1-dioxide, preparedaccording to the procedure described in the Journal of Organic Chemistry(1990), 55(4), 1254-61.

Suitable N-phosphonyl imine oxygen transfer catalysts include, but arenot limited to,[R-(E)]-N-[(2-chloro-5-nitrophenyl)methylene]-P-phenyl-P-(2,4,6-trimethylphenyl)-phosphinicamide, which can be made according to the procedures described in theJournal of the Chemical Society, Chemical Communications (1994), (22),2569-70.

Suitable N-acyl imine oxygen transfer catalysts include, but are notlimited to, [N(E)]-N-(phenylmethylene)acetamide, which can be madeaccording to the procedures described in Polish Journal of Chemistry(2003), 77(5), 577-590.

Suitable thiadiazole dioxide oxygen transfer catalysts include but arenot limited to, 3-methyl-4-phenyl-1,2,5-thiadiazole 1,1-dioxide, whichcan be made according to the procedures described in U.S. Pat. No.5,753,599 (Column 9, Ex. 2).

Suitable perfluoroimine oxygen transfer catalysts include, but are notlimited to,(Z)-2,2,3,3,4,4,4-heptafluoro-N-(nonafluorobutyl)butanimidoyl fluoride,which can be made according to the procedures described in TetrahedronLetters (1994), 35(34), 6329-30.

Suitable cyclic sugar ketone oxygen transfer catalysts include, but arenot limited to,1,2:4,5-di-O-isopropylidene-D-erythro-2,3-hexodiuro-2,6-pyranose asprepared in U.S. Pat. No. 6,649,085 (Column 12, Ex. 1).

Preferably, the bleach catalyst comprises an iminium and/or carbonylfunctional group and is typically capable of forming an oxaziridiniumand/or dioxirane functional group upon acceptance of an oxygen atom,especially upon acceptance of an oxygen atom from a peroxyacid and/orsalt thereof. Preferably, the bleach catalyst comprises an oxaziridiniumfunctional group and/or is capable of forming an oxaziridiniumfunctional group upon acceptance of an oxygen atom, especially uponacceptance of an oxygen atom from a peroxyacid and/or salt thereof.Preferably, the bleach catalyst comprises a cyclic iminium functionalgroup, preferably wherein the cyclic moiety has a ring size of from fiveto eight atoms (including the nitrogen atom), preferably six atoms.Preferably, the bleach catalyst comprises an aryliminium functionalgroup, preferably a bi-cyclic aryliminium functional group, preferably a3,4-dihydroisoquinolinium functional group. Typically, the iminefunctional group is a quaternary imine functional group and is typicallycapable of forming a quaternary oxaziridinium functional group uponacceptance of an oxygen atom, especially upon acceptance of an oxygenatom from a peroxyacid and/or salt thereof. In another aspect, thedetergent composition comprises a bleach component having a log Pm_(o/w)no greater than 0, no greater than −0.5, no greater than −1.0, nogreater than −1.5, no greater than −2.0, no greater than −2.5, nogreater than −3.0, or no greater than −3.5. The method for determininglog P_(o/w) is described in more detail below.

Typically, the bleach ingredient is capable of generating a bleachingspecies having a X_(SO) of from 0.01 to 0.30, from 0.05 to 0.25, or from0.10 to 0.20. The method for determining X_(SO) is described in moredetail below. For example, bleaching ingredients having anisoquinolinium structure are capable of generating a bleaching speciesthat has an oxaziridinium structure. In this example, the X_(SO) is thatof the oxaziridinium bleaching species.

Preferably, the bleach catalyst has a chemical structure correspondingto the following chemical formula:

wherein: n and m are independently from 0 to 4, preferably n and m areboth 0; each R¹ is independently selected from a substituted orunsubstituted radical selected from the group consisting of hydrogen,alkyl, cycloalkyl, aryl, fused aryl, heterocyclic ring, fusedheterocyclic ring, nitro, halo, cyano, sulphonato, alkoxy, keto,carboxylic, and carboalkoxy radicals; and any two vicinal R¹substituents may combine to form a fused aryl, fused carbocyclic orfused heterocyclic ring; each R² is independently selected from asubstituted or unsubstituted radical independently selected from thegroup consisting of hydrogen, hydroxy, alkyl, cycloalkyl, alkaryl, aryl,aralkyl, alkylenes, heterocyclic ring, alkoxys, arylcarbonyl groups,carboxyalkyl groups and amide groups; any R² may be joined together withany other of R² to form part of a common ring; any geminal R² maycombine to form a carbonyl; and any two R² may combine to form asubstituted or unsubstituted fused unsaturated moiety; R³ is a C₁ to C₂₀substituted or unsubstituted alkyl; R⁴ is hydrogen or the moietyQ_(t)-A, wherein: Q is a branched or unbranched alkylene, t=0 or 1 and Ais an anionic group selected from the group consisting of OSO₃ ⁻³, SO₃⁻, CO₂ ⁻, OCO₂ ⁻, OPO₃ ²⁻, OPO₃H⁻ and OPO₂ ⁻; R⁵ is hydrogen or themoiety —CR¹¹R¹²—Y—G_(b)—Y_(c)—[(CR⁹R¹⁰)_(y)—O]_(k)—R⁸, wherein: each Yis independently selected from the group consisting of O, S, N—H, orN—R⁸; and each R⁸ is independently selected from the group consisting ofalkyl, aryl and heteroaryl, said moieties being substituted orunsubstituted, and whether substituted or unsubstituted said moietieshaving less than 21 carbons; each G is independently selected from thegroup consisting of CO, SO₂, SO, PO and PO₂; R⁹ and R¹⁰ areindependently selected from the group consisting of H and C₁-C₄ alkyl;R¹¹ and R¹² are independently selected from the group consisting of Hand alkyl, or when taken together may join to form a carbonyl; b=0 or 1;c can=0 or 1, but c must=0 if b=0; y is an integer from 1 to 6; k is aninteger from 0 to 20; R⁶ is H, or an alkyl, aryl or heteroaryl moiety;said moieties being substituted or unsubstituted; and X, if present, isa suitable charge balancing counterion, preferably X is present when R⁴is hydrogen, suitable X, include but are not limited to: chloride,bromide, sulphate, methosulphate, sulphonate, p-toluenesulphonate,borontetraflouride and phosphate.

In one embodiment of the present invention, the bleach catalyst has astructure corresponding to general formula below:

wherein R¹³ is a branched alkyl group containing from three to 24 carbonatoms (including the branching carbon atoms) or a linear alkyl groupcontaining from one to 24 carbon atoms; preferably R¹³ is a branchedalkyl group containing from eight to 18 carbon atoms or linear alkylgroup containing from eight to eighteen carbon atoms; preferably R¹³ isselected from the group consisting of 2-propylheptyl, 2-butyloctyl,2-pentylnonyl, 2-hexyldecyl, n-dodecyl, n-tetradecyl, n-hexadecyl,n-octadecyl, iso-nonyl, iso-decyl, iso-tridecyl and iso-pentadecyl;preferably R¹³ is selected from the group consisting of 2-butyloctyl,2-pentylnonyl, 2-hexyldecyl, iso-tridecyl and iso-pentadecyl.

Preferably the bleach component comprises a source of peracid inaddition to bleach catalyst, particularly organic bleach catalyst. Thesource of peracid may be selected from (a) pre-formed peracid; (b)percarbonate, perborate or persulfate salt (hydrogen peroxide source)preferably in combination with a bleach activator; and (c) perhydrolaseenzyme and an ester for forming peracid in situ in the presence of waterin a textile or hard surface treatment step.

When present, the peracid and/or bleach activator is generally presentin the composition in an amount of from 0.1 to 60 wt %, from 0.5 to 40wt % or from 0.6 to 10 wt % based on the composition. One or morehydrophobic peracids or precursors thereof may be used in combinationwith one or more hydrophilic peracid or precursor thereof.

The amounts of hydrogen peroxide source and peracid or bleach activatormay be selected such that the molar ratio of available oxygen (from theperoxide source) to peracid is from 1:1 to 35:1, or 2:1 to 10:1.

(6) Metal-containing Bleach Catalysts—The bleach component may beprovided by a catalytic metal complex. One type of metal-containingbleach catalyst is a catalyst system comprising a transition metalcation of defined bleach catalytic activity, such as copper, iron,titanium, ruthenium, tungsten, molybdenum, or manganese cations, anauxiliary metal cation having little or no bleach catalytic activity,such as zinc or aluminum cations, and a sequestrate having definedstability constants for the catalytic and auxiliary metal cations,particularly ethylenediaminetetraacetic acid,ethylenediaminetetra(methylenephosphonic acid) and water-soluble saltsthereof. Such catalysts are disclosed in U.S. Pat. No. 4,430,243.Preferred catalysts are described in WO09/839406, U.S. Pat. No.6,218,351 and WO00/012667. Particularly preferred are transition metalcatalyst or ligands therefore that are cross-bridged polydentate N-donorligands.

If desired, the compositions herein can be catalyzed by means of amanganese compound. Such compounds and levels of use are well known inthe art and include, e.g., the manganese-based catalysts disclosed inU.S. Pat. No. 5,576,282.

Cobalt bleach catalysts useful herein are known, and are described e.g.in U.S. Pat. Nos. 5,597,936; 5,595,967. Such cobalt catalysts arereadily prepared by known procedures, such as taught e.g. in U.S. Pat.Nos. 5,597,936 and 5,595,967.

Compositions herein may also suitably include a transition metal complexof ligands such as bispidones (U.S. Pat. No. 7,501,389) and/ormacropolycyclic rigid ligands—abbreviated as “MRLs”. As a practicalmatter, and not by way of limitation, the compositions and processesherein can be adjusted to provide on the order of at least one part perhundred million of the active MRL species in the aqueous washing medium,and will typically provide from 0.005 to 25 ppm, from 0.05 to 10 ppm, orfrom 0.1 to 5 ppm, of the MRL in the wash liquor.

Suitable transition-metals in the instant transition-metal bleachcatalyst include e.g. manganese, iron and chromium. Suitable MRLsinclude 5,12-diethyl-1,5,8,12-tetraazabicyclo[6.6.2]hexadecane. Suitabletransition metal MRLs are readily prepared by known procedures, such astaught e.g. in U.S. Pat. No. 6,225,464 and WO00/32601.

(7) Photobleaches—suitable photobleaches include e.g. sulfonated zincphthalocyanine sulfonated aluminium phthalocyanines, xanthene dyes andmixtures thereof. Preferred bleach components for use in the presentcompositions of the invention comprise a hydrogen peroxide source,bleach activator and/or organic peroxyacid, optionally generated in situby the reaction of a hydrogen peroxide source and bleach activator, incombination with a bleach catalyst. Preferred bleach components comprisebleach catalysts, preferably organic bleach catalysts, as describedabove.

Particularly preferred bleach components are the bleach catalysts inparticular the organic bleach catalysts.

Exemplary bleaching systems are also described, e.g. in WO2007/087258,WO2007/087244, WO2007/087259 and WO2007/087242.

Fabric Hueing Agents—The composition may comprise a fabric hueing agent.Suitable fabric hueing agents include dyes, dye-clay conjugates, andpigments. Suitable dyes include small molecule dyes and polymeric dyes.Suitable small molecule dyes include small molecule dyes selected fromthe group consisting of dyes falling into the Color Index (C.I.)classifications of Direct Blue, Direct Red, Direct Violet, Acid Blue,Acid Red, Acid Violet, Basic Blue, Basic Violet and Basic Red, ormixtures thereof.

In another aspect, suitable small molecule dyes include small moleculedyes selected from the group consisting of Color Index (Society of Dyersand Colorists, Bradford, UK) numbers Direct Violet 9, Direct Violet 35,Direct Violet 48, Direct Violet 51, Direct Violet 66, Direct Violet 99,Direct Blue 1, Direct Blue 71, Direct Blue 80, Direct Blue 279, Acid Red17, Acid Red 73, Acid Red 88, Acid Red 150, Acid Violet 15, Acid Violet17, Acid Violet 24, Acid Violet 43, Acid Red 52, Acid Violet 49, AcidViolet 50, Acid Blue 15, Acid Blue 17, Acid Blue 25, Acid Blue 29, AcidBlue 40, Acid Blue 45, Acid Blue 75, Acid Blue 80, Acid Blue 83, AcidBlue 90 and Acid Blue 113, Acid Black 1, Basic Violet 1, Basic Violet 3,Basic Violet 4, Basic Violet 10, Basic Violet 35, Basic Blue 3, BasicBlue 16, Basic Blue 22, Basic Blue 47, Basic Blue 66, Basic Blue 75,Basic Blue 159 and mixtures thereof. In another aspect, suitable smallmolecule dyes include small molecule dyes selected from the groupconsisting of Color Index (Society of Dyers and Colorists, Bradford, UK)numbers Acid Violet 17, Acid Violet 43, Acid Red 52, Acid Red 73, AcidRed 88, Acid Red 150, Acid Blue 25, Acid Blue 29, Acid Blue 45, AcidBlue 113, Acid Black 1, Direct Blue 1, Direct Blue 71, Direct Violet 51and mixtures thereof. In another aspect, suitable small molecule dyesinclude small molecule dyes selected from the group consisting of ColorIndex (Society of Dyers and Colorists, Bradford, UK) numbers Acid Violet17, Direct Blue 71, Direct Violet 51, Direct Blue 1, Acid Red 88, AcidRed 150, Acid Blue 29, Acid Blue 113 or mixtures thereof.

Suitable polymeric dyes include polymeric dyes selected from the groupconsisting of polymers containing conjugated chromogens (dye-polymerconjugates) and polymers with chromogens co-polymerized into thebackbone of the polymer and mixtures thereof.

In another aspect, suitable polymeric dyes include polymeric dyesselected from the group consisting of fabric-substantive colorants soldunder the name of Liquitint® (Milliken), dye-polymer conjugates formedfrom at least one reactive dye and a polymer selected from the groupconsisting of polymers comprising a moiety selected from the groupconsisting of a hydroxyl moiety, a primary amine moiety, a secondaryamine moiety, a thiol moiety and mixtures thereof. In still anotheraspect, suitable polymeric dyes include polymeric dyes selected from thegroup consisting of Liquitint® Violet CT, carboxymethyl cellulose (CMC)conjugated with a reactive blue, reactive violet or reactive red dyesuch as CMC conjugated with C.I. Reactive Blue 19, sold by Megazyme,Wicklow, Ireland under the product name AZO-CM-CELLULOSE, product codeS-ACMC, alkoxylated triphenyl-methane polymeric colorants, alkoxylatedthiophene polymeric colorants, and mixtures thereof.

Preferred hueing dyes include the whitening agents found in WO08/87497.These whitening agents may be characterized by the following structure(I):

wherein R₁ and R₂ can independently be selected from:

a) [(CH₂CR′HO)_(x)(CH₂CR″HO)_(y)H]

wherein R′ is selected from the group consisting of H, CH₃,CH₂O(CH₂CH₂O)_(z)H, and mixtures thereof; wherein R″ is selected fromthe group consisting of H, CH₂O(CH₂CH₂O)_(z)H, and mixtures thereof;wherein x+y≤5; wherein y≥1; and wherein z=0 to 5;

b) R₁=alkyl, aryl or aryl alkyl and R₂=[(CH₂CR′HO)_(x)(CH₂CR″HO)_(y)H]wherein R′ is selected from the group consisting of H, CH3,CH₂O(CH₂CH₂O)_(z)H, and mixtures thereof; wherein R″ is selected fromthe group consisting of H, CH₂O(CH₂CH₂O)_(z)H, and mixtures thereof;wherein x+y≤10; wherein y≥1; and wherein z=0 to 5;

c) R₁=[CH₂CH₂(OR₃)CH₂OR₄] and R₂=[CH₂CH₂(OR₃)CH₂OR₄] wherein R₃ isselected from the group consisting of H, (CH₂CH₂O)_(z)H, and mixturesthereof; and wherein z=0 to 10;

wherein R₄ is selected from the group consisting of (C₁-C₁₆)alkyl, arylgroups, and mixtures thereof; and

d) wherein R₁ and R₂ can independently be selected from the aminoaddition product of styrene oxide, glycidyl methyl ether, isobutylglycidyl ether, isopropylglycidyl ether, t-butyl glycidyl ether,2-ethylhexylgycidyl ether, and glycidylhexadecyl ether, followed by theaddition of from 1 to 10 alkylene oxide units.

A preferred whitening agent of the present invention may becharacterized by the following structure (II):

wherein R′ is selected from the group consisting of H, CH₃,CH₂O(CH₂CH₂O)_(z)H, and mixtures thereof; wherein R″ is selected fromthe group consisting of H, CH₂O(CH₂CH₂O)_(z)H, and mixtures thereof;wherein x+y≤5; wherein y≥1; and wherein z=0 to 5.

A further preferred whitening agent of the present invention may becharacterized by the following structure (III):

typically comprising a mixture having a total of 5 EO groups. Suitablepreferred molecules are those in Structure I having the followingpendant groups in “part a” above.

TABLE 1 R1 R2 R′ R″ X y R′ R″ x y A H H 3 1 H H 0 1 B H H 2 1 H H 1 1 c= b H H 1 1 H H 2 1 d = a H H 0 1 H H 3 1Further whitening agents of use include those described in US2008/34511(Unilever). A preferred agent is “Violet 13”.

Suitable dye clay conjugates include dye clay conjugates selected fromthe group comprising at least one cationic/basic dye and a smectiteclay, and mixtures thereof. In another aspect, suitable dye clayconjugates include dye clay conjugates selected from the groupconsisting of one cationic/basic dye selected from the group consistingof C.I. Basic Yellow 1 through 108, C.I. Basic Orange 1 through 69, C.I.Basic Red 1 through 118, C.I. Basic Violet 1 through 51, C.I. Basic Blue1 through 164, C.I. Basic Green 1 through 14, C.I. Basic Brown 1 through23, CI Basic Black 1 through 11, and a clay selected from the groupconsisting of Montmorillonite clay, Hectorite clay, Saponite clay andmixtures thereof. In still another aspect, suitable dye clay conjugatesinclude dye clay conjugates selected from the group consisting of:Montmorillonite Basic Blue B7 C.I. 42595 conjugate, MontmorilloniteBasic Blue B9 C.I. 52015 conjugate, Montmorillonite Basic Violet V3 C.I.42555 conjugate, Montmorillonite Basic Green G1 C.I. 42040 conjugate,Montmorillonite Basic Red R1 C.I. 45160 conjugate, Montmorillonite C.I.Basic Black 2 conjugate, Hectorite Basic Blue B7 C.I. 42595 conjugate,Hectorite Basic Blue B9 C.I. 52015 conjugate, Hectorite Basic Violet V3C.I. 42555 conjugate, Hectorite Basic Green G1 C.I. 42040 conjugate,Hectorite Basic Red R1 C.I. 45160 conjugate, Hectorite C.I. Basic Black2 conjugate, Saponite Basic Blue B7 C.I. 42595 conjugate, Saponite BasicBlue B9 C.I. 52015 conjugate, Saponite Basic Violet V3 C.I. 42555conjugate, Saponite Basic Green G1 C.I. 42040 conjugate, Saponite BasicRed R1 C.I. 45160 conjugate, Saponite C.I. Basic Black 2 conjugate andmixtures thereof.

Suitable pigments include pigments selected from the group consisting offlavanthrone, indanthrone, chlorinated indanthrone containing from 1 to4 chlorine atoms, pyranthrone, dichloropyranthrone,monobromodichloropyranthrone, dibromodichloropyranthrone,tetrabromopyranthrone, perylene-3,4,9,10-tetracarboxylic acid diimide,wherein the imide groups may be unsubstituted or substituted byC1-C3-alkyl or a phenyl or heterocyclic radical, and wherein the phenyland heterocyclic radicals may additionally carry substituents which donot confer solubility in water, anthrapyrimidinecarboxylic acid amides,violanthrone, isoviolanthrone, dioxazine pigments, copper phthalocyaninewhich may contain up to 2 chlorine atoms per molecule, polychloro-copperphthalocyanine or polybromochloro-copper phthalocyanine containing up to14 bromine atoms per molecule and mixtures thereof.

In another aspect, suitable pigments include pigments selected from thegroup consisting of Ultramarine Blue (CA. Pigment Blue 29), UltramarineViolet (CA. Pigment Violet 15) and mixtures thereof.

The aforementioned fabric hueing agents can be used in combination (anymixture of fabric hueing agents can be used). Suitable hueing agents aredescribed in more detail in U.S. Pat. No. 7,208,459. Preferred levels ofdye in compositions of the invention are 0.00001 to 0.5 wt %, or 0.0001to 0.25 wt %. The concentration of dyes preferred in water for thetreatment and/or cleaning step is from 1 ppb to 5 ppm, 10 ppb to 5 ppmor 20 ppb to 5 ppm. In preferred compositions, the concentration ofsurfactant will be from 0.2 to 3 g/l.

Encapsulates—The composition may comprise an encapsulate. In one aspect,an encapsulate comprising a core, a shell having an inner and outersurface, said shell encapsulating said core.

In one aspect of said encapsulate, said core may comprise a materialselected from the group consisting of perfumes; brighteners; dyes;insect repellants; silicones; waxes; flavors; vitamins; fabric softeningagents; skin care agents in one aspect, paraffins; enzymes;anti-bacterial agents; bleaches; sensates; and mixtures thereof; andsaid shell may comprise a material selected from the group consisting ofpolyethylenes; polyamides; polyvinylalcohols, optionally containingother co-monomers; polystyrenes; polyisoprenes; polycarbonates;polyesters; polyacrylates; aminoplasts, in one aspect said aminoplastmay comprise a polyureas, polyurethane, and/or polyureaurethane, in oneaspect said polyurea may comprise polyoxymethyleneurea and/or melamineformaldehyde; polyolefins; polysaccharides, in one aspect saidpolysaccharide may comprise alginate and/or chitosan; gelatin; shellac;epoxy resins; vinyl polymers; water insoluble inorganics; silicone; andmixtures thereof.

In one aspect of said encapsulate, said core may comprise perfume.

In one aspect of said encapsulate, said shell may comprise melamineformaldehyde and/or cross linked melamine formaldehyde.

In a one aspect, suitable encapsulates may comprise a core material anda shell, said shell at least partially surrounding said core material,is disclosed. At least 75%, 85% or 90% of said encapsulates may have afracture strength of from 0.2 to 10 MPa, from 0.4 to 5 MPa, from 0.6 to3.5 MPa, or from 0.7 to 3 MPa; and a benefit agent leakage of from 0 to30%, from 0 to 20%, or from 0 to 5%.

In one aspect, at least 75%, 85% or 90% of said encapsulates may have aparticle size from 1 to 80 microns, from 5 to 60 microns, from 10 to 50microns, or from 15 to 40 microns.

In one aspect, at least 75%, 85% or 90% of said encapsulates may have aparticle wall thickness from 30 to 250 nm, from 80 to 180 nm, or from100 to 160 nm.

In one aspect, said encapsulates' core material may comprise a materialselected from the group consisting of a perfume raw material and/oroptionally a material selected from the group consisting of vegetableoil, including neat and/or blended vegetable oils including castor oil,coconut oil, cottonseed oil, grape oil, rapeseed, soybean oil, corn oil,palm oil, linseed oil, safflower oil, olive oil, peanut oil, coconutoil, palm kernel oil, castor oil, lemon oil and mixtures thereof; estersof vegetable oils, esters, including dibutyl adipate, dibutyl phthalate,butyl benzyl adipate, benzyl octyl adipate, tricresyl phosphate,trioctyl phosphate and mixtures thereof; straight or branched chainhydrocarbons, including those straight or branched chain hydrocarbonshaving a boiling point of greater than about 80° C.; partiallyhydrogenated terphenyls, dialkyl phthalates, alkyl biphenyls, includingmonoisopropylbiphenyl, alkylated naphthalene, includingdipropylnaphthalene, petroleum spirits, including kerosene, mineral oiland mixtures thereof; aromatic solvents, including benzene, toluene andmixtures thereof; silicone oils; and mixtures thereof.

In one aspect, said encapsulates' wall material may comprise a suitableresin including the reaction product of an aldehyde and an amine,suitable aldehydes include, formaldehyde. Suitable amines includemelamine, urea, benzoguanamine, glycoluril, and mixtures thereof.Suitable melamines include methylol melamine, methylated methylolmelamine, imino melamine and mixtures thereof. Suitable ureas includedimethylol urea, methylated dimethylol urea, urea-resorcinol, andmixtures thereof.

In one aspect, suitable formaldehyde scavengers may be employed with theencapsulates e.g. in a capsule slurry and/or added to a compositionbefore, during or after the encapsulates are added to such composition.Suitable capsules may be made by the following teaching ofUS2008/0305982; and/or US2009/0247449.

In a preferred aspect the composition can also comprise a depositionaid, preferably consisting of the group comprising cationic or nonionicpolymers. Suitable polymers include cationic starches, cationichydroxyethylcellulose, polyvinylformaldehyde, locust bean gum, mannans,xyloglucans, tamarind gum, polyethyleneterephthalate and polymerscontaining dimethylaminoethyl methacrylate, optionally with one ormonomers selected from the group comprising acrylic acid and acrylamide.

Perfumes—In one aspect the composition comprises a perfume thatcomprises one or more perfume raw materials selected from the groupconsisting of 1,1′-oxybis-2-propanol; 1,4-cyclohexanedicarboxylic acid,diethyl ester; (ethoxymethoxy)cyclododecane; 1,3-nonanediol,monoacetate; (3-methylbutoxy)acetic acid, 2-propenyl ester; beta-methylcyclododecaneethanol;2-methyl-3-[(1,7,7-trimethylbicyclo[2.2.1]hept-2-yl)oxy]-1-propanol;oxacyclohexadecan-2-one; alpha-methyl-benzenemethanol acetate;trans-3-ethoxy-1,1,5-trimethylcyclohexane;4-(1,1-dimethylethyl)cyclohexanol acetate;dodecahydro-3a,6,6,9a-tetramethylnaphtho[2,1-b]furan; beta-methylbenzenepropanal; beta-methyl-3-(1-methylethyl)benzenepropanal;4-phenyl-2-butanone; 2-methylbutanoic acid, ethyl ester; benzaldehyde;2-methylbutanoic acid, 1-methylethyl ester;dihydro-5-pentyl-2(3H)furanone;(2E)-1-(2,6,6-trimethyl-2-cyclohexen-1-yl)-2-buten-1-one; dodecanal;undecanal; 2-ethyl-alpha, alpha-dimethylbenzenepropanal; decanal; alpha,alpha-dimethylbenzeneethanol acetate; 2-(phenylmethylene)octanal;2-[[3-[4-(1,1-dimethylethyl)phenyl]-2-methylpropylidene]amino]benzoicacid, methyl ester; 1-(2,6,6-trimethyl-3-cyclohexen-1-yl)-2-buten-1-one;2-pentylcyclopentanone; 3-oxo-2-pentyl cyclopentaneacetic acid, methylester; 4-hydroxy-3-methoxybenzaldehyde; 3-ethoxy-4-hydroxybenzaldehyde;2-heptylcyclopentanone; 1-(4-methylphenyl)ethanone;(3E)-4-(2,6,6-trimethyl-1-cyclohexen-1-yl)-3-buten-2-one;(3E)-4-(2,6,6-trimethyl-2-cyclohexen-1-yl)-3-buten-2-one;benzeneethanol; 2H-1-benzopyran-2-one; 4-methoxybenzaldehyde;10-undecenal; propanoic acid, phenylmethyl ester;beta-methylbenzenepentanol; 1,1-diethoxy-3,7-dimethyl-2,6-octadiene;alpha, alpha-dimethylbenzeneethanol;(2E)-1-(2,6,6-trimethyl-1-cyclohexen-1-yl)-2-buten-1-one; acetic acid,phenylmethyl ester; cyclohexanepropanoic acid, 2-propenyl ester;hexanoic acid, 2-propenyl ester; 1,2-dimethoxy-4-(2-propenyl)benzene;1,5-dimethyl-bicyclo[3.2.1]octan-8-one oxime;4-(4-hydroxy-4-methylpentyl)-3-cyclohexene-1-carboxaldehyde;3-buten-2-ol; 2-[[[2,4(or3,5)-dimethyl-3-cyclohexen-1-yl]methylene]amino]benzoic acid, methylester; 8-cyclohexadecen-1-one; methyl ionone; 2,6-dimethyl-7-octen-2-ol;2-methoxy-4-(2-propenyl)phenol; (2E)-3,7-dimethyl-2,6-Octadien-1-ol;2-hydroxy-Benzoic acid, (3Z)-3-hexenyl ester; 2-tridecenenitrile;4-(2,2-dimethyl-6-methylenecyclohexyl)-3-methyl-3-buten-2-one;tetrahydro-4-methyl-2-(2-methyl-1-propenyl)-2H-pyran; Acetic acid,(2-methylbutoxy)-, 2-propenyl ester; Benzoic acid,2-hydroxy-,3-methylbutyl ester; 2-Buten-1-one,1-(2,6,6-trimethyl-1-cyclohexen-1-yl)-, (Z)-; Cyclopentanecarboxylicacid, 2-hexyl-3-oxo-, methyl ester; Benzenepropanal,4-ethyl-.alpha.,.alpha.-dimethyl-; 3-Cyclohexene-1-carboxaldehyde,3-(4-hydroxy-4-methylpentyl)-; Ethanone,1-(2,3,4,7,8,8a-hexahydro-3,6,8,8-tetramethyl-1H-3a,7-methanoazulen-5-yl)-,[3R-(3.alpha.,3a.beta.,7.beta.,8a.alpha.)]-; Undecanal,2-methyl-2H-Pyran-2-one, 6-butyltetrahydro-; Benzenepropanal,4-(1,1-dimethylethyl)-.alpha.-methyl-; 2(3H)-Furanone, 5-heptyldihydro-;Benzoic acid, 2-[(7-hydroxy-3,7-dimethyloctylidene)amino]-, methyl;Benzoic acid, 2-hydroxy-, phenylmethyl ester; Naphthalene, 2-methoxy-;2-Cyclopenten-1-one, 2-hexyl-; 2(3H)-Furanone, 5-hexyldihydro-;Oxiranecarboxylic acid, 3-methyl-3-phenyl-, ethyl ester;2-Oxabicyclo[2.2.2]octane, 1,3,3-trimethyl-; Benzenepentanol,.gamma.-methyl-; 3-Octanol, 3,7-dimethyl-;3,7-dimethyl-2,6-octadienenitrile; 3,7-dimethyl-6-octen-1-ol; Terpineolacetate; 2-methyl-6-methylene-7-Octen-2-ol, dihydro derivative;3a,4,5,6,7,7a-hexahydro-4,7-Methano-1H-inden-6-ol propanoate;3-methyl-2-buten-1-ol acetate; (Z)-3-Hexen-1-ol acetate;2-ethyl-4-(2,2,3-trimethyl-3-cyclopenten-1-yl)-2-buten-1-ol;4-(octahydro-4,7-methano-5H-inden-5-ylidene)-butanal;3-2,4-dimethyl-cyclohexene-1-carboxaldehyde;1-(1,2,3,4,5,6,7,8-octahydro-2,3,8,8-tetramethyl-2-naphthalenyl)-ethanone;2-hydroxy-benzoic acid, methyl ester; 2-hydroxy-benzoic acid, hexylester; 2-phenoxy-ethanol; 2-hydroxy-benzoic acid, pentyl ester;2,3-heptanedione; 2-hexen-1-ol; 6-Octen-2-ol, 2,6-dimethyl-; damascone(alpha, beta, gamma or delta or mixtures thereof),4,7-Methano-1H-inden-6-ol, 3a,4,5,6,7,7a-hexahydro-, acetate;9-Undecenal; 8-Undecenal; Isocyclocitral; Ethanone,1-(1,2,3,5,6,7,8,8a-octahydro-2,3,8,8-tetramethyl-2-naphthalenyl)-;3-Cyclohexene-1-carboxaldehyde, 3,5-dimethyl-;3-Cyclohexene-1-carboxaldehyde, 2,4-dimethyl-; 1,6-Octadien-3-ol,3,7-dimethyl-; 1,6-Octadien-3-ol, 3,7-dimethyl-, acetate; Lilial(p-t-Bucinal), and Cyclopentanone,2-[2-(4-methyl-3-cyclohexen-1-yl)propyl]- and1-methyl-4-(1-methylethenyl)cyclohexene and mixtures thereof.

In one aspect the composition may comprise an encapsulated perfumeparticle comprising either a water-soluble hydroxylic compound ormelamine-formaldehyde or modified polyvinyl alcohol. In one aspect theencapsulate comprises (a) an at least partially water-soluble solidmatrix comprising one or more water-soluble hydroxylic compounds,preferably starch; and (b) a perfume oil encapsulated by the solidmatrix.

In a further aspect the perfume may be pre-complexed with a polyamine,preferably a polyethylenimine so as to form a Schiff base.

Polymers—The composition may comprise one or more polymers. Examples arecarboxymethylcellulose, poly(vinyl-pyrrolidone), poly (ethylene glycol),poly(vinyl alcohol), poly(vinylpyridine-N-oxide), poly(vinylimidazole),polycarboxylates such as polyacrylates, maleic/acrylic acid copolymersand lauryl methacrylate/acrylic acid co-polymers.

The composition may comprise one or more amphiphilic cleaning polymerssuch as the compound having the following general structure:bis((C₂H₅O)(C₂H₄O)n)(CH₃)—N⁺—C_(x)H_(2x)—N⁺—(CH₃)-bis((C₂H₅O)(C₂H₄O)n),wherein n=from 20 to 30, and x=from 3 to 8, or sulphated or sulphonatedvariants thereof.

The composition may comprise amphiphilic alkoxylated grease cleaningpolymers which have balanced hydrophilic and hydrophobic properties suchthat they remove grease particles from fabrics and surfaces. Specificembodiments of the amphiphilic alkoxylated grease cleaning polymers ofthe present invention comprise a core structure and a plurality ofalkoxylate groups attached to that core structure. These may comprisealkoxylated polyalkylenimines, preferably having an inner polyethyleneoxide block and an outer polypropylene oxide block.

Alkoxylated polycarboxylates such as those prepared from polyacrylatesare useful herein to provide additional grease removal performance. Suchmaterials are described in WO91/08281 and PCT90/01815. Chemically, thesematerials comprise polyacrylates having one ethoxy side-chain per every7-8 acrylate units. The side-chains are of the formula —(CH₂CH₂O)_(m)(CH₂)_(n)CH₃ wherein m is 2-3 and n is 6-12. The side-chains areester-linked to the polyacrylate “backbone” to provide a “comb” polymertype structure. The molecular weight can vary, but is typically in therange of 2000 to 50,000. Such alkoxylated polycarboxylates can comprisefrom 0.05 wt % to 10 wt % of the compositions herein.

The isoprenoid-derived surfactants of the present invention, and theirmixtures with other cosurfactants and other adjunct ingredients, areparticularly suited to be used with an amphilic graft co-polymer,preferably the amphilic graft co-polymer comprises (i) polyethyeleneglycol backbone; and (ii) and at least one pendant moiety selected frompolyvinyl acetate, polyvinyl alcohol and mixtures thereof. A preferredamphilic graft co-polymer is Sokalan HP22, supplied from BASF. Suitablepolymers include random graft copolymers, preferably a polyvinyl acetategrafted polyethylene oxide copolymer having a polyethylene oxidebackbone and multiple polyvinyl acetate side chains. The molecularweight of the polyethylene oxide backbone is preferably 6000 and theweight ratio of the polyethylene oxide to polyvinyl acetate is 40 to 60and no more than 1 grafting point per 50 ethylene oxide units.

Carboxylate polymer—The composition of the present invention may alsoinclude one or more carboxylate polymers such as a maleate/acrylaterandom copolymer or polyacrylate homopolymer. In one aspect, thecarboxylate polymer is a polyacrylate homopolymer having a molecularweight of from 4,000 to 9,000 Da, or from 6,000 to 9,000 Da.

Soil release polymer—The composition of the present invention may alsoinclude one or more soil release polymers having a structure as definedby one of the following structures (I), (II) or (III):

(I) —[(OCHR¹—CHR²)_(a)—O—OC—Ar—CO—]_(d)

(II) —[(OCHR³—CHR⁴)_(b)—O—OC-sAr—CO—]_(e)

(III) —[(OCHR⁵—CHR⁶)_(c)—OR⁷]_(f)

wherein:

-   a, b and c are from 1 to 200;-   d, e and f are from 1 to 50;-   Ar is a 1,4-substituted phenylene;-   sAr is 1,3-substituted phenylene substituted in position 5 with    SO₃Me;-   Me is Li, K, Mg/2, Ca/2, Al/3, ammonium, mono-, di-, tri-, or    tetraalkylammonium wherein the alkyl groups are C₁-C₁₈ alkyl or    C₂-C₁₀ hydroxyalkyl, or mixtures thereof;-   R¹, R², R³, R⁴, R⁵ and R⁶ are independently selected from H or    C₁-C₁₈n- or iso-alkyl; and-   R⁷ is a linear or branched C₁-C₁₈ alkyl, or a linear or branched    C₂-C₃₀ alkenyl, or a cycloalkyl group with 5 to 9 carbon atoms, or a    C₈-C₃₀ aryl group, or a C₆-C₃₀ arylalkyl group.

Suitable soil release polymers are polyester soil release polymers suchas Repel-o-tex polymers, including Repel-o-tex, SF-2 and SRP6 suppliedby Rhodia. Other suitable soil release polymers include Texcarepolymers, including Texcare SRA100, SRA300, SRN100, SRN170, SRN240,SRN300 and SRN325 supplied by Clariant. Other suitable soil releasepolymers are Marloquest polymers, such as Marloquest SL supplied bySasol.

Cellulosic polymer—The composition of the present invention may alsoinclude one or more cellulosic polymers including those selected fromalkyl cellulose, alkyl alkoxyalkyl cellulose, carboxyalkyl cellulose,alkyl carboxyalkyl cellulose. In one aspect, the cellulosic polymers areselected from the group comprising carboxymethyl cellulose, methylcellulose, methyl hydroxyethyl cellulose, methyl carboxymethylcellulose, and mixures thereof. In one aspect, the carboxymethylcellulose has a degree of carboxymethyl substitution from 0.5 to 0.9 anda molecular weight from 100,000 to 300,000 Da.

Enzymes—The composition may comprise one or more enzymes which providecleaning performance and/or fabric care benefits. Examples of suitableenzymes include, but are not limited to, hemicellulases, peroxidases,proteases, cellulases, xylanases, lipases, phospholipases, esterases,cutinases, pectinases, mannanases, pectate lyases, keratinases,reductases, oxidases, phenoloxidases, lipoxygenases, ligninases,pullulanases, tannases, pentosanases, malanases, ß-glucanases,arabinosidases, hyaluronidase, chondroitinase, laccase, chlorophyllases,amylases, or mixtures thereof. A typical combination is an enzymecocktail that may comprise e.g. a protease and lipase in conjunctionwith amylase. When present in a composition, the aforementionedadditional enzymes may be present at levels from 0.00001 to 2 wt %, from0.0001 to 1 wt % or from 0.001 to 0.5 wt % enzyme protein by weight ofthe composition.

In general the properties of the selected enzyme(s) should be compatiblewith the selected detergent, (i.e., pH-optimum, compatibility with otherenzymatic and non-enzymatic ingredients, etc.), and the enzyme(s) shouldbe present in effective amounts.

In one aspect preferred enzymes would include a cellulase. Suitablecellulases include those of bacterial or fungal origin. Chemicallymodified or protein engineered mutants are included. Suitable cellulasesinclude cellulases from the genera Bacillus, Pseudomonas, Humicola,Fusarium, Thielavia, Acremonium, e.g., the fungal cellulases producedfrom Humicola insolens, Myceliophthora thermophila and Fusariumoxysporum disclosed in U.S. Pat. Nos. 4,435,307, 5,648,263, 5,691,178,5,776,757 and WO89/09259.

Especially suitable cellulases are the alkaline or neutral cellulaseshaving colour care benefits. Examples of such cellulases are cellulasesdescribed in EP0495257, EP0531372, WO96/11262, WO96/29397, WO98/08940.Other examples are cellulase variants such as those described inWO94/07998, EP0531315, U.S. Pat. Nos. 5,457,046, 5,686,593, 5,763,254,WO95/24471, WO98/12307 and PCT/DK98/00299.

Commercially available cellulases include Celluzyme™, and Carezyme™(Novozymes A/S), Clazinase™, and Puradax HA™ (Genencor InternationalInc.), and KAC-500(B)™ (Kao Corporation).

In one aspect preferred enzymes would include a protease. Suitableproteases include those of bacterial, fungal, plant, viral or animalorigin e.g. vegetable or microbial origin. Microbial origin ispreferred. Chemically modified or protein engineered mutants areincluded. It may be an alkaline protease, such as a serine protease or ametalloprotease. A serine protease may for example be of the 51 family,such as trypsin, or the S8 family such as subtilisin. A metalloproteasesprotease may for example be a thermolysin from e.g. family M4 or othermetalloprotease such as those from M5, M7 or M8 families.

The term “subtilases” refers to a sub-group of serine protease accordingto Siezen et al., Protein Engng. 4 (1991) 719-737 and Siezen et al.Protein Science 6 (1997) 501-523. Serine proteases are a subgroup ofproteases characterized by having a serine in the active site, whichforms a covalent adduct with the substrate. The subtilases may bedivided into 6 sub-divisions, i.e. the Subtilisin family, the Thermitasefamily, the Proteinase K family, the Lantibiotic peptidase family, theKexin family and the Pyrolysin family.

Examples of subtilases are those derived from Bacillus such as Bacilluslentus, B. alkalophilus, B. subtilis, B. amyloliquefaciens, Bacilluspumilus and Bacillus gibsonii described in; U.S. Pat. No. 7,262,042 andWO09/021867, and subtilisin lentus, subtilisin Novo, subtilisinCarlsberg, Bacillus licheniformis, subtilisin BPN′, subtilisin 309,subtilisin 147 and subtilisin 168 described in WO89/06279 and proteasePD138 described in (WO93/18140). Other useful proteases may be thosedescribed in WO92/175177, WO01/016285, WO02/026024 and WO02/016547.Examples of trypsin-like proteases are trypsin (e.g. of porcine orbovine origin) and the Fusarium protease described in WO89/06270,WO94/25583 and WO05/040372, and the chymotrypsin proteases derived fromCellumonas described in WO05/052161 and WO05/052146.

A further preferred protease is the alkaline protease from Bacilluslentus DSM 5483, as described for example in WO95/23221, and variantsthereof which are described in WO92/21760, WO95/23221, EP1921147 andEP1921148.

Examples of metalloproteases are the neutral metalloprotease asdescribed in WO07/044993 (Genencor Int.) such as those derived fromBacillus amyloliquefaciens. Examples of useful proteases are thevariants described in: WO92/19729, WO96/034946, WO98/20115, WO98/20116,WO99/011768, WO01/44452, WO03/006602, WO04/03186, WO04/041979,WO07/006305, WO11/036263, WO11/036264, especially the variants withsubstitutions in one or more of the following positions: 3, 4, 9, 15,27, 36, 57, 68, 76, 87, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,106, 118, 120, 123, 128, 129, 130, 160, 167, 170, 194, 195, 199, 205,206, 217, 218, 222, 224, 232, 235, 236, 245, 248, 252 and 274 using theBPN′ numbering. More preferred the subtilase variants may comprise themutations: S3T, V4I, S9R, A15T, K27R, *36D, V68A, N76D, N87S,R, *97E,A98S, S99G,D,A, S99AD, S101G,M,R S103A, V104I,Y,N, S106A, G118V,R,H120D,N, N123S, S128L, P129Q, S130A, G160D, Y167A, R170S, A194P, G195E,V199M, V205I, L217D, N218D, M222S, A232V, K235L, Q236H, Q245R, N252K,T274A (using BPN′ numbering).

Suitable commercially available protease enzymes include those soldunder the trade names Alcalase®, Blaze®; Duralase™, Durazym™, Relase®,Relase® Ultra, Savinase®, Savinase® Ultra, Primase®, Polarzyme®,Kannase®, Liquanase®, Liquanase® Ultra, Ovozyme®, Coronase®, Coronase®Ultra,Neutrase®, Everlase® and Esperase® all could be sold as Ultra® orEvity® (Novozymes NS), those sold under the tradename Maxatase®,Maxacal®, Maxapem®, Purafect®, Purafect Prime®, Preferenz™, PurafectMA®, Purafect Ox®, Purafect OxP®, Puramax®, Properase®, Effectenz™,FN2®, FN3®, FN4®, Excellase®, Opticlean® and Optimase® (Danisco/DuPont),Axapem™ (Gist-Brocases N. V.), BLAP (sequence shown in FIG. 29 of U.S.Pat. No. 5,352,604) and variants hereof (Henkel AG) and KAP (Bacillusalkalophilus subtilisin) from Kao.

In one aspect preferred enzymes would include an amylase. Suitableamylases may be an alpha-amylase or a glucoamylase and may be ofbacterial or fungal origin. Chemically modified or protein engineeredmutants are included. Amylases include, for example, alpha-amylasesobtained from Bacillus, e.g., a special strain of Bacilluslicheniformis, described in more detail in GB1296839.

Suitable amylases include amylases having SEQ ID NO: 3 in WO95/10603 orvariants having 90% sequence identity to SEQ ID NO: 3 thereof. Preferredvariants are described in WO94/02597, WO94/18314, WO97/43424 and SEQ IDNO: 4 of WO99/019467, such as variants with substitutions in one or moreof the following positions: 15, 23, 105, 106, 124, 128, 133, 154, 156,178, 179, 181, 188, 190, 197, 201, 202, 207, 208, 209, 211, 243, 264,304, 305, 391, 408, and 444.

Different suitable amylases include amylases having SEQ ID NO: 6 inWO02/010355 or variants thereof having 90% sequence identity to SEQ IDNO: 6. Preferred variants of SEQ ID NO: 6 are those having a deletion inpositions 181 and 182 and a substitution in position 193.

Other amylases which are suitable are hybrid alpha-amylase comprisingresidues 1-33 of the alpha-amylase derived from B. amyloliquefaciensshown in SEQ ID NO: 6 of WO2006/066594 and residues 36-483 of the B.licheniformis alpha-amylase shown in SEQ ID NO: 4 of WO2006/066594 orvariants having 90% sequence identity thereof. Preferred variants ofthis hybrid alpha-amylase are those having a substitution, a deletion oran insertion in one of more of the following positions: G48, T49, G107,H156, A181, N190, M197, I201, A209 and Q264. Most preferred variants ofthe hybrid alpha-amylase comprising residues 1-33 of the alpha-amylasederived from B. amyloliquefaciens shown in SEQ ID NO: 6 of WO2006/066594and residues 36-483 of SEQ ID NO: 4 are those having the substitutions:

M197T;

H156Y+A181T+N190F+A209V+Q264S; or

G48A+T49I+G107A+H156Y+A181T+N190F+I201F+A209V+Q264S.

Further amylases which are suitable are amylases having SEQ ID NO: 6 inWO99/019467 or variants thereof having 90% sequence identity to SEQ IDNO: 6. Preferred variants of SEQ ID NO: 6 are those having asubstitution, a deletion or an insertion in one or more of the followingpositions: R181, G182, H183, G184, N195, I206, E212, E216 and K269.Particularly preferred amylases are those having deletion in positionsR181 and G182, or positions H183 and G184.

Additional amylases which can be used are those having SEQ ID NO: 1, SEQID NO: 3, SEQ ID NO: 2 or SEQ ID NO: 7 of WO96/023873 or variantsthereof having 90% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQID NO: 3 or SEQ ID NO: 7. Preferred variants of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3 or SEQ ID NO: 7 are those having a substitution, adeletion or an insertion in one or more of the following positions: 140,181, 182, 183, 184, 195, 206, 212, 243, 260, 269, 304 and 476. Morepreferred variants are those having a deletion in positions 181 and 182or positions 183 and 184. Most preferred amylase variants of SEQ ID NO:1, SEQ ID NO: 2 or SEQ ID NO: 7 are those having a deletion in positions183 and 184 and a substitution in one or more of positions 140, 195,206, 243, 260, 304 and 476.

Other amylases which can be used are amylases having SEQ ID NO: 2 ofWO08/153815, SEQ ID NO: 10 in WO01/66712 or variants thereof having 90%sequence identity to SEQ ID NO: 2 of WO08/153815 or 90% sequenceidentity to SEQ ID NO: 10 in WO01/66712. Preferred variants of SEQ IDNO: 10 in WO01/66712 are those having a substitution, a deletion or aninsertion in one of more of the following positions: 176, 177, 178, 179,190, 201, 207, 211 and 264.

Further suitable amylases are amylases having SEQ ID NO: 2 ofWO09/061380 or variants having 90% sequence identity to SEQ ID NO: 2thereof. Preferred variants of SEQ ID NO: 2 are those having atruncation of the C-terminus and/or a substitution, a deletion or aninsertion in one of more of the following positions: Q87, Q98, S125,N128, T131, T165, K178, R180, S181, T182, G183, M201, F202, N225, S243,N272, N282, Y305, R309, D319, Q320, Q359, K444 and G475. More preferredvariants of SEQ ID NO: 2 are those having the substitution in one ofmore of the following positions: Q87E,R, Q98R, S125A, N128C, T131I,T165I, K178L, T182G, M201L, F202Y, N225E,R, N272E,R, S243Q,A,E,D, Y305R,R309A, Q320R, Q359E, K444E and G475K and/or deletion in position R180and/or S181 or of T182 and/or G183. Most preferred amylase variants ofSEQ ID NO: 2 are those having the substitutions:

N128C+K178L+T182G+Y305R+G475K;

N128C+K178L+T182G+F202Y+Y305R+D319T+G475K;

S125A+N128C+K178L+T182G+Y305R+G475K; or

S125A+N128C+T131I+T165I+K178L+T182G+Y305R+G475K wherein the variants areC-terminally truncated and optionally further comprises a substitutionat position 243 and/or a deletion at position 180 and/or position 181.

Other suitable amylases are the alpha-amylase having SEQ ID NO: 12 inWO01/66712 or a variant having at least 90% sequence identity to SEQ IDNO: 12. Preferred amylase variants are those having a substitution, adeletion or an insertion in one of more of the following positions ofSEQ ID NO: 12 in WO01/66712: R28, R118, N174; R181, G182, D183, G184,G186, W189, N195, M202, Y298, N299, K302, S303, N306, R310, N314; R320,H324, E345, Y396, R400, W439, R444, N445, K446, Q449, R458, N471, N484.Particular preferred amylases include variants having a deletion of D183and G184 and having the substitutions R118K, N195F, R320K and R458K, anda variant additionally having substitutions in one or more positionselected from the group: M9, G149, G182, G186, M202, T257, Y295, N299,M323, E345 and A339, most preferred a variant that additionally hassubstitutions in all these positions.

Other examples are amylase variants such as those described inWO2011/098531, WO2013/001078 and WO2013/001087.

Commercially available amylases are Duramyl™, Termamyl™, TermamylUltra™, Fungamyl™, Ban™, Stainzyme™, Stainzyme Plus™, Amplify®,Supramyl™, Natalase™, Liquozyme X and BAN™ (from Novozymes NS), KEMZYM®AT 9000 Biozym Biotech Trading GmbH Wehlistrasse 27b A-1200 WienAustria, and Rapidase™, Purastar™/Effectenz™, Powerase, Preferenz S100,Preferenx S110, ENZYSIZE®, OPTISIZE HT PLUS®, and PURASTAR OXAM®(Danisco/DuPont) and KAM® (Kao).

Suitable lipases and cutinases include those of bacterial or fungalorigin. Chemically modified or protein engineered mutant enzymes areincluded. Examples include lipase from Thermomyces, e.g. from T.lanuginosus (previously named Humicola lanuginosa) as described inEP258068 and EP305216, cutinase from Humicola, e.g. H. insolens(WO96/13580), lipase from strains of Pseudomonas (some of these nowrenamed to Burkholderia), e.g. P. alcaligenes or P. pseudoalcaligenes(EP218272), P. cepacia (EP331376), P. sp. strain SD705 (WO95/06720 &WO96/27002), P. wisconsinensis (WO96/12012), GDSL-type Streptomyceslipases (WO10/065455), cutinase from Magnaporthe grisea (WO10/107560),cutinase from Pseudomonas mendocina (U.S. Pat. No. 5,389,536), lipasefrom Thermobifida fusca (WO11/084412, WO13/033318), Geobacillusstearothermophilus lipase (WO11/084417), lipase from Bacillus subtilis(WO11/084599), and lipase from Streptomyces griseus (WO11/150157) and S.pristinaespiralis (WO12/137147).

Other examples are lipase variants such as those described in EP407225,WO92/05249, WO94/01541, WO94/25578, WO95/14783, WO95/30744, WO95/35381,WO95/22615, WO96/00292, WO97/04079, WO97/07202, WO00/34450, WO00/60063,WO01/92502, WO07/87508 and WO09/109500.

Preferred commercial lipase products include Lipolase™, Lipex™; Lipolex™and Lipoclean™ (Novozymes NS), Lumafast (originally from Genencor) andLipomax (originally from Gist-Brocades).

Still other examples are lipases sometimes referred to asacyltransferases or perhydrolases, e.g. acyltransferases with homologyto Candida antarctica lipase A (WO10/111143), acyltransferase fromMycobacterium smegmatis (WO05/56782), perhydrolases from the CE 7 family(WO09/67279), and variants of the M. smegmatis perhydrolase inparticular the S54V variant used in the commercial product Gentle PowerBleach from Huntsman Textile Effects Pte Ltd (WO10/100028).

In one aspect, other preferred enzymes include microbial-derivedendoglucanases exhibiting endo-beta-1,4-glucanase activity (EC3.2.1.4),including a bacterial polypeptide endogenous to a member of the genusBacillus which has a sequence of at least 90%, 94%, 97% or 99% identityto the amino acid sequence SEQ ID NO:2 in U.S. Pat. No. 7,141,403 andmixtures thereof. Suitable endoglucanases are sold under the tradenamesCelluclean® and Whitezyme® (Novozymes).

Other preferred enzymes include pectate lyases sold under the tradenamesPectawash®, Pectaway®, Xpect® and mannanases sold under the tradenamesMannaway® (Novozymes), and Purabrite® (Danisco/DuPont).

The detergent enzyme(s) may be included in a detergent composition byadding separate additives containing one or more enzymes, or by adding acombined additive comprising all of these enzymes. A detergent additiveof the invention, i.e., a separate additive or a combined additive, canbe formulated, for example, as granulate, liquid, slurry, etc. Preferreddetergent additive formulations are granulates, in particularnon-dusting granulates, liquids, in particular stabilized liquids, orslurries.

Non-dusting granulates may be produced, e.g. as disclosed in U.S. Pat.Nos. 4,106,991 and 4,661,452 and may optionally be coated by methodsknown in the art. Examples of waxy coating materials are poly(ethyleneoxide) products (polyethyleneglycol, PEG) with mean molar weights of1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethyleneoxide units; ethoxylated fatty alcohols in which the alcohol containsfrom 12 to 20 carbon atoms and in which there are 15 to 80 ethyleneoxide units; fatty alcohols; fatty acids; and mono- and di- andtriglycerides of fatty acids. Examples of film-forming coating materialssuitable for application by fluid bed techniques are given in GB1483591.Liquid enzyme preparations may, for instance, be stabilized by adding apolyol such as propylene glycol, a sugar or sugar alcohol, lactic acidor boric acid according to established methods. Protected enzymes may beprepared according to the method disclosed in EP238216.

Dye Transfer Inhibiting Agents—The compositions of the present inventionmay also include one or more dye transfer inhibiting agents. Suitablepolymeric dye transfer inhibiting agents include, but are not limitedto, polyvinylpyrrolidone polymers, polyamine N-oxide polymers,copolymers of N-vinylpyrrolidone and N-vinylimidazole,polyvinyloxazolidones and polyvinylimidazoles or mixtures thereof. Whenpresent in a composition, the dye transfer inhibiting agents may bepresent at levels from 0.0001 to 10 wt %, from 0.01 to 5 wt % or from0.1 to 3 wt %.

Brighteners—The compositions of the present invention can also containadditional components that may tint articles being cleaned, such asfluorescent brighteners.

The composition may comprise C.I. fluorescent brightener 260 inalpha-crystalline form having the following structure:

In one aspect, the brightener is a cold water soluble brightener, suchas the C.I. fluorescent brightener 260 in alpha-crystalline form. In oneaspect the brightener is predominantly in alpha-crystalline form, whichmeans that typically at least 50 wt %, at least 75 wt %, at least 90 wt%, at least 99 wt %, or even substantially all, of the C.I. fluorescentbrightener 260 is in alpha-crystalline form.

The brightener is typically in micronized particulate form, having aweight average primary particle size of from 3 to 30 micrometers, from 3micrometers to 20 micrometers, or from 3 to 10 micrometers.

The composition may comprise C.I. fluorescent brightener 260 inbeta-crystalline form, and the weight ratio of: (i) C.I. fluorescentbrightener 260 in alpha-crystalline form, to (ii) C.I. fluorescentbrightener 260 in beta-crystalline form may be at least 0.1, or at least0.6. BE680847 relates to a process for making 0.1 fluorescent brightener260 in alpha-crystalline form.

Commercial optical brighteners which may be useful in the presentinvention can be classified into subgroups, which include, but are notnecessarily limited to, derivatives of stilbene, pyrazoline, coumarin,carboxylic acid, methinecyanines, dibenzothiophene-5,5-dioxide, azoles,5- and 6-membered-ring heterocycles, and other miscellaneous agents.Examples of such brighteners are disclosed in “The Production andApplication of Fluorescent Brightening Agents”, M. Zahradnik, Publishedby John Wiley & Sons, New York (1982). Specific nonlimiting examples ofoptical brighteners which are useful in the present compositions arethose identified in U.S. Pat. Nos. 4,790,856 and 3,646,015.

A further suitable brightener has the structure below:

Suitable fluorescent brightener levels include lower levels of from 0.01wt %, from 0.05 wt %, from 0.1 wt % or from 0.2 wt % to upper levels of0.5 wt % or 0.75 wt %.

In one aspect the brightener may be loaded onto a clay to form aparticle. Silicate salts—The compositions of the present invention canalso contain silicate salts, such as sodium or potassium silicate. Thecomposition may comprise of from 0 wt % to less than 10 wt % silicatesalt, to 9 wt %, or to 8 wt %, or to 7 wt %, or to 6 wt %, or to 5 wt %,or to 4 wt %, or to 3 wt %, or even to 2 wt %, and from above 0 wt %, orfrom 0.5 wt %, or from 1 wt % silicate salt. A suitable silicate salt issodium silicate.

Dispersants—The compositions of the present invention can also containdispersants. Suitable water-soluble organic materials include the homo-or co-polymeric acids or their salts, in which the polycarboxylic acidcomprises at least two carboxyl radicals separated from each other bynot more than two carbon atoms.

Enzyme Stabilizers—Enzymes for use in compositions can be stabilized byvarious techniques. The enzymes employed herein can be stabilized by thepresence of water-soluble sources of calcium and/or magnesium ions.Examples of conventional stabilizing agents are, e.g. a polyol such aspropylene glycol or glycerol, a sugar or sugar alcohol, a peptidealdehyde, lactic acid, boric acid, or a boric acid derivative, e.g. anaromatic borate ester, or a phenyl boronic acid derivative such as4-formylphenyl boronic acid, and the composition may be formulated asdescribed in, for example, WO92/19709 and WO92/19708 In case of aqueouscompositions comprising protease, a reversible protease inhibitor, suchas a boron compound including borate, 4-formyl phenylboronic acid,phenylboronic acid and derivatives thereof, or compounds such as calciumformate, sodium formate and 1,2-propane diol can be added to furtherimprove stability. The peptide aldehyde may be of the formula B₂—B₁—B₀—Rwherein: R is hydrogen, CH₃, CX₃, CHX₂, or CH₂X, wherein X is a halogenatom; B₀ is a phenylalanine residue with an OH substituent at thep-position and/or at the m-position; B₁ is a single amino acid residue;and B₂ consists of one or more amino acid residues, optionallycomprising an N-terminal protection group. Preferred peptide aldehydesinclude but are not limited to: Z-RAY-H, Ac-GAY-H, Z-GAY-H, Z-GAL-H,Z-GAF-H, Z-GAV-H, Z-RVY-H, Z-LVY-H, Ac-LGAY-H, Ac-FGAY-H, Ac-YGAY-H,Ac-FGVY-H or Ac-WLVY-H, where Z is benzyloxycarbonyl and Ac is acetyl.

Solvents—Suitable solvents include water and other solvents such aslipophilic fluids. Examples of suitable lipophilic fluids includesiloxanes, other silicones, hydrocarbons, glycol ethers, glycerinederivatives such as glycerine ethers, perfluorinated amines,perfluorinated and hydrofluoroether solvents, low-volatilitynonfluorinated organic solvents, diol solvents, otherenvironmentally-friendly solvents and mixtures thereof.

Structurant/Thickeners—Structured liquids can either be internallystructured, whereby the structure is formed by primary ingredients (e.g.surfactant material) and/or externally structured by providing a threedimensional matrix structure using secondary ingredients (e.g. polymers,clay and/or silicate material). The composition may comprise astructurant, from 0.01 to 5 wt %, or from 0.1 to 2.0 wt %. Thestructurant is typically selected from the group consisting ofdiglycerides and triglycerides, ethylene glycol distearate,microcrystalline cellulose, cellulose-based materials, microfibercellulose, hydrophobically modified alkali-swellable emulsions such asPolygel W30 (3VSigma), biopolymers, xanthan gum, gellan gum, andmixtures thereof. A suitable structurant includes hydrogenated castoroil, and non-ethoxylated derivatives thereof. A suitable structurant isdisclosed in U.S. Pat. No. 6,855,680. Such structurants have athread-like structuring system having a range of aspect ratios. Othersuitable structurants and the processes for making them are described inWO10/034736.

Conditioning Agents—The composition of the present invention may includea high melting point fatty compound. The high melting point fattycompound useful herein has a melting point of 25° C. or higher, and isselected from the group consisting of fatty alcohols, fatty acids, fattyalcohol derivatives, fatty acid derivatives, and mixtures thereof. Suchcompounds of low melting point are not intended to be included in thissection. Non-limiting examples of the high melting point compounds arefound in International Cosmetic Ingredient Dictionary, Fifth Edition,1993, and CTFA Cosmetic Ingredient Handbook, Second Edition, 1992.

The high melting point fatty compound is included in the composition atalevel of from 0.1 to 40 wt %, from 1 to 30 wt %, from 1.5 to 16 wt %,from 1.5 to 8 wt % in view of providing improved conditioning benefitssuch as slippery feel during the application to wet hair, softness andmoisturized feel on dry hair.

The compositions of the present invention may contain a cationicpolymer. Concentrations of the cationic polymer in the compositiontypically range from 0.05 to 3 wt %, from 0.075 to 2.0 wt %, or from 0.1to 1.0 wt %. Suitable cationic polymers will have cationic chargedensities of at least 0.5 meq/gm, at least 0.9 meq/gm, at least 1.2meq/gm, at least 1.5 meq/gm, or less than 7 meq/gm, and less than 5meq/gm, at the pH of intended use of the composition, which pH willgenerally range from pH3 to pH9, or between pH4 and pH8. Herein,“cationic charge density” of a polymer refers to the ratio of the numberof positive charges on the polymer to the molecular weight of thepolymer. The average molecular weight of such suitable cationic polymerswill generally be between 10,000 and 10 million, between 50,000 and 5million, or between 100,000 and 3 million.

Suitable cationic polymers for use in the compositions of the presentinvention contain cationic nitrogen-containing moieties such asquaternary ammonium or cationic protonated amino moieties. Any anioniccounterions can be used in association with the cationic polymers solong as the polymers remain soluble in water, in the composition, or ina coacervate phase of the composition, and so long as the counterionsare physically and chemically compatible with the essential componentsof the composition or do not otherwise unduly impair compositionperformance, stability or aesthetics. Nonlimiting examples of suchcounterions include halides (e.g., chloride, fluoride, bromide, iodide),sulfate and methylsulfate.

Nonlimiting examples of such polymers are described in the CTFA CosmeticIngredient Dictionary, 3rd edition, edited by Estrin, Crosley, andHaynes, (The Cosmetic, Toiletry, and Fragrance Association, Inc.,Washington, D. C. (1982)).

Other suitable cationic polymers for use in the composition includepolysaccharide polymers, cationic guar gum derivatives, quaternarynitrogen-containing cellulose ethers, synthetic polymers, copolymers ofetherified cellulose, guar and starch. When used, the cationic polymersherein are either soluble in the composition or are soluble in a complexcoacervate phase in the composition formed by the cationic polymer andthe anionic, amphoteric and/or zwitterionic surfactant componentdescribed hereinbefore. Complex coacervates of the cationic polymer canalso be formed with other charged materials in the composition. Suitablecationic polymers are described in U.S. Pat. Nos. 3,962,418; 3,958,581;and US2007/0207109.

The composition of the present invention may include a nonionic polymeras a conditioning agent. Polyalkylene glycols having a molecular weightof more than 1000 are useful herein. Useful are those having thefollowing general formula:

wherein R⁹⁵ is selected from the group consisting of H, methyl, andmixtures thereof. Conditioning agents, and in particular silicones, maybe included in the composition. The conditioning agents useful in thecompositions of the present invention typically comprise a waterinsoluble, water dispersible, non-volatile, liquid that formsemulsified, liquid particles. Suitable conditioning agents for use inthe composition are those conditioning agents characterized generally assilicones (e.g., silicone oils, cationic silicones, silicone gums, highrefractive silicones, and silicone resins), organic conditioning oils(e.g., hydrocarbon oils, polyolefins, and fatty esters) or combinationsthereof, or those conditioning agents which otherwise form liquid,dispersed particles in the aqueous surfactant matrix herein. Suchconditioning agents should be physically and chemically compatible withthe essential components of the composition, and should not otherwiseunduly impair composition stability, aesthetics or performance.

The concentration of the conditioning agent in the composition should besufficient to provide the desired conditioning benefits. Suchconcentration can vary with the conditioning agent, the conditioningperformance desired, the average size of the conditioning agentparticles, the type and concentration of other components, and otherlike factors.

The concentration of the silicone conditioning agent typically rangesfrom 0.01 to 10 wt %. Non-limiting examples of suitable siliconeconditioning agents, and optional suspending agents for the silicone,are described in U.S. Reissue Pat. No. 34,584; U.S. Pat. Nos. 5,104,646;5,106,609; 4,152,416; 2,826,551; 3,964,500; 4,364,837; 6,607,717;6,482,969; 5,807,956; 5,981,681; 6,207,782; 7,465,439; 7,041,767;7,217,777; US2007/0286837A1; US2005/0048549A1; US2007/0041929A1;GB849433; DE10036533, which are all incorporated herein by reference;Chemistry and Technology of Silicones, New York: Academic Press (1968);General Electric Silicone Rubber Product Data Sheets SE 30, SE 33, SE 54and SE 76; Silicon Compounds, Petrarch Systems, Inc. (1984); and inEncyclopedia of Polymer Science and Engineering, vol. 15, 2d ed., pp204-308, John Wiley & Sons, Inc. (1989).

The compositions of the present invention may also comprise from 0.05 to3 wt % of at least one organic conditioning oil as the conditioningagent, either alone or in combination with other conditioning agents,such as the silicones (described herein). Suitable conditioning oilsinclude hydrocarbon oils, polyolefins, and fatty esters. Also suitablefor use in the compositions herein are the conditioning agents describedin U.S. Pat. Nos. 5,674,478 and 5750122 or in U.S. Pat. Nos. 4,529,586;4,507,280; 4,663,158; 4,197,865; 4,217,914; 4,381,919; and 4,422,853.

Hygiene and malodour—The compositions of the present invention may alsocomprise one or more of zinc ricinoleate, thymol, quaternary ammoniumsalts such as Bardac®, polyethylenimines (such as Lupasol® from BASF)and zinc complexes thereof, silver and silver compounds, especiallythose designed to slowly release Ag⁺ or nano-silver dispersions.

Probiotics—The compositions may comprise probiotics such as thosedescribed in WO09/043709.

Suds Boosters—If high sudsing is desired, suds boosters such as theC₁₀-C₁₆ alkanolamides or C₁₀-C₁₄ alkyl sulphates can be incorporatedinto the compositions, typically at 1 to 10 wt % levels. The C₁₀-C₁₄monoethanol and diethanol amides illustrate a typical class of such sudsboosters. Use of such suds boosters with high sudsing adjunctsurfactants such as the amine oxides, betaines and sultaines noted aboveis also advantageous. If desired, water-soluble magnesium and/or calciumsalts such as MgCl₂, MgSO₄, CaCl₂, CaSO₄ and the like, can be added atlevels of, typically, 0.1 to 2 wt %, to provide additional suds and toenhance grease removal performance.

Suds Suppressors—Compounds for reducing or suppressing the formation ofsuds can be incorporated into the compositions of the present invention.Suds suppression can be of particular importance in the so-called “highconcentration cleaning process” as described in U.S. Pat. Nos. 4,489,455and 4,489,574, and in front-loading-style washing machines. A widevariety of materials may be used as suds suppressors, and sudssuppressors are well known to those skilled in the art. See e.g. KirkOthmer Encyclopedia of Chemical Technology, Third Edition, Volume 7, p.430-447 (John Wiley & Sons, Inc., 1979). Examples of suds supressorsinclude monocarboxylic fatty acid and soluble salts therein, highmolecular weight hydrocarbons such as paraffin, fatty acid esters (e.g.,fatty acid triglycerides), fatty acid esters of monovalent alcohols,aliphatic C₁₈-C₄₀ ketones (e.g., stearone), N-alkylated amino triazines,waxy hydrocarbons preferably having a melting point below about 100° C.,silicone suds suppressors, and secondary alcohols. Suds supressors aredescribed in U.S. Pat. Nos. 2,954,347; 4,265,779; 4,265,779; 3,455,839;3,933,672; 4,652,392; 4,978,471; 4,983,316; 5,288,431; 4,639,489;4,749,740; 4,798,679; 4,075,118; EP89307851.9; EP150872; and DOS2,124,526.

For any detergent compositions to be used in automatic laundry washingmachines, suds should not form to the extent that they overflow thewashing machine. Suds suppressors, when utilized, are preferably presentin a “suds suppressing amount. By “suds suppressing amount” is meantthat the formulator of the composition can select an amount of this sudscontrolling agent that will sufficiently control the suds to result in alow-sudsing laundry detergent for use in automatic laundry washingmachines.

The compositions herein will generally comprise from 0 to 10 wt % ofsuds suppressor. When utilized as suds suppressors, monocarboxylic fattyacids, and salts therein, will be present typically in amounts up to 5wt %. Preferably, from 0.5 to 3 wt % of fatty monocarboxylate sudssuppressor is utilized. Silicone suds suppressors are typically utilizedin amounts up to 2.0 wt %, although higher amounts may be used.Monostearyl phosphate suds suppressors are generally utilized in amountsranging from 0.1 to 2 wt %. Hydrocarbon suds suppressors are typicallyutilized in amounts ranging from 0.01 to 5.0 wt %, although higherlevels can be used. The alcohol suds suppressors are typically used at0.2 to 3 wt %.

The compositions herein may have a cleaning activity over a broad rangeof pH. In certain embodiments the compositions have cleaning activityfrom pH4 to pH11.5. In other embodiments, the compositions are activefrom pH6 to pH11, from pH7 to pH11, from pH8 to pH11, from pH9 to pH11,or from pH10 to pH11.5.

The compositions herein may have cleaning activity over a wide range oftemperatures, e.g., from 10° C. or lower to 90° C. Preferably thetemperature will be below 50° C. or 40° C. or even 30° C. In certainembodiments, the optimum temperature range for the compositions is from10° C. to 20° C., from 15° C. to 25° C., from 15° C. to 30° C., from 20°C. to 30° C., from 25° C. to 35° C., from 30° C. to 40° C., from 35° C.to 45° C., or from 40° C. to 50° C.

Form of the Composition

The compositions described herein are advantageously employed forexample, in laundry applications, hard surface cleaning, dishwashingapplications, as well as cosmetic applications such as dentures, teeth,hair and skin. The compositions of the invention are in particular solidor liquid cleaning and/or treatment compositions. In one aspect theinvention relates to a composition, wherein the form of the compositionis selected from the group consisting of a regular, compact orconcentrated liquid; a gel; a paste; a soap bar; a regular or acompacted powder; a granulated solid; a homogenous or a multilayertablet with two or more layers (same or different phases); a pouchhaving one or more compartments; a single or a multi-compartment unitdose form; or any combination thereof.

The form of the composition may separate the components physically fromeach other in compartments such as e.g. water dissolvable pouches or indifferent layers of tablets. Thereby negative storage interactionbetween components can be avoided. Different dissolution profiles ofeach of the compartments can also give rise to delayed dissolution ofselected components in the wash solution.

Pouches can be configured as single or multicompartments. It can be ofany form, shape and material which is suitable for hold the composition,e.g. without allowing the release of the composition to release of thecomposition from the pouch prior to water contact. The pouch is madefrom water soluble film which encloses an inner volume. Said innervolume can be divided into compartments of the pouch. Preferred filmsare polymeric materials preferably polymers which are formed into a filmor sheet. Preferred polymers, copolymers or derivates thereof areselected polyacrylates, and water soluble acrylate copolymers, methylcellulose, carboxy methyl cellulose, sodium dextrin, ethyl cellulose,hydroxyethyl cellulose, hydroxypropyl methyl cellulose, malto dextrin,poly methacrylates, most preferably polyvinyl alcohol copolymers and,hydroxypropyl methyl cellulose (HPMC). Preferably the level of polymerin the film for example PVA is at least about 60%. Preferred averagemolecular weight will typically be about 20,000 to about 150,000. Filmscan also be of blended compositions comprising hydrolytically degradableand water soluble polymer blends such as polylactide and polyvinylalcohol (known under the Trade reference M8630 as sold by MonoSol LLC,Indiana, USA) plus plasticisers like glycerol, ethylene glycerol,propylene glycol, sorbitol and mixtures thereof. The pouches cancomprise a solid laundry cleaning composition or part components and/ora liquid cleaning composition or part components separated by the watersoluble film. The compartment for liquid components can be different incomposition than compartments containing solids (US2009/0011970 A1).

Water-Soluble Film—The compositions of the present invention may also beencapsulated within a water-soluble film. Preferred film materials arepreferably polymeric materials. The film material can e.g. be obtainedby casting, blow-moulding, extrusion or blown extrusion of the polymericmaterial, as known in the art. Preferred polymers, copolymers orderivatives thereof suitable for use as pouch material are selected frompolyvinyl alcohols, polyvinyl pyrrolidone, polyalkylene oxides,acrylamide, acrylic acid, cellulose, cellulose ethers, cellulose esters,cellulose amides, polyvinyl acetates, polycarboxylic acids and salts,polyaminoacids or peptides, polyamides, polyacrylamide, copolymers ofmaleic/acrylic acids, polysaccharides including starch and gelatine,natural gums such as xanthum and carragum. More preferred polymers areselected from polyacrylates and water-soluble acrylate copolymers,methylcellulose, carboxymethylcellulose sodium, dextrin, ethylcellulose,hydroxyethyl cellulose, hydroxypropyl methylcellulose, maltodextrin,polymethacrylates, and most preferably selected from polyvinyl alcohols,polyvinyl alcohol copolymers and hydroxypropyl methyl cellulose (HPMC),and combinations thereof. Preferably, the level of polymer in the pouchmaterial, e.g. a PVA polymer, is at least 60 wt %. The polymer can haveany weight average molecular weight, preferably from about 1.000 to1.000.000, from about 10.000 to 300.000, from about 20.000 to 150.000.Mixtures of polymers can also be used as the pouch material.

Naturally, different film material and/or films of different thicknessmay be employed in making the compartments of the present invention. Abenefit in selecting different films is that the resulting compartmentsmay exhibit different solubility or release characteristics.

Preferred film materials are PVA films known under the MonoSol tradereference M8630, M8900, H8779 and those described in U.S. Pat. Nos.6,166,117 and 6,787,512 and PVA films of corresponding solubility anddeformability characteristics.

The film material herein can also comprise one or more additiveingredients. For example, it can be beneficial to add plasticisers, e.g.glycerol, ethylene glycol, diethyleneglycol, propylene glycol, sorbitoland mixtures thereof. Other additives include functional detergentadditives to be delivered to the wash water, e.g. organic polymericdispersants, etc.

Processes of Making the Compositions

The compositions of the present invention can be formulated into anysuitable form and prepared by any process chosen by the formulator,non-limiting examples of which are described in Applicants' examples andin U.S. Pat. No. 4,990,280; US20030087791A1; US20030087790A1;US20050003983A1; US20040048764A1; U.S. Pat. Nos. 4,762,636; 6,291,412;US20050227891A1; EP1070115A2; U.S. Pat. Nos. 5,879,584; 5,691,297;5,574,005; 5,569,645; 5,565,422; 5,516,448; 5,489,392; 5,486,303 all ofwhich are incorporated herein by reference. The compositions of theinvention or prepared according to the invention comprise cleaningand/or treatment composition including, but not limited to, compositionsfor treating fabrics, hard surfaces and any other surfaces in the areaof fabric and home care, including: air care including air freshenersand scent delivery systems, car care, dishwashing, fabric conditioning(including softening and/or freshening), laundry detergency, laundry andrinse additive and/or care, hard surface cleaning and/or treatmentincluding floor and toilet bowl cleaners, granular or powder-formall-purpose or “heavy-duty” washing agents, especially cleaningdetergents; liquid, gel or paste-form all-purpose washing agents,especially the so-called heavy-duty liquid types; liquid fine-fabricdetergents; hand dishwashing agents or light duty dishwashing agents,especially those of the high-foaming type; machine dishwashing agents,including the various tablet, granular, liquid and rinse-aid types forhousehold and institutional use: car or carpet shampoos, bathroomcleaners including toilet bowl cleaners; as well as cleaning auxiliariessuch as bleach additives and “stain-stick” or pre-treat types,substrate-laden compositions such as dryer added sheets. Preferred arecompositions and methods for cleaning and/or treating textiles and/orhard surfaces, most preferably textiles. The compositions are preferablycompositions used in a pre-treatment step or main wash step of a washingprocess, most preferably for use in textile washing step.

As used herein, the term “fabric and/or hard surface cleaning and/ortreatment composition” is a subset of cleaning and treatmentcompositions that includes, unless otherwise indicated, granular orpowder-form all-purpose or “heavy-duty” washing agents, especiallycleaning detergents; liquid, gel or paste-form all-purpose washingagents, especially the so-called heavy-duty liquid types; liquidfine-fabric detergents; hand dishwashing agents or light dutydishwashing agents, especially those of the high-foaming type; machinedishwashing agents, including the various tablet, granular, liquid andrinse-aid types for household and institutional use; liquid cleaning anddisinfecting agents, car or carpet shampoos, bathroom cleaners includingtoilet bowl cleaners; fabric conditioning compositions includingsoftening and/or freshening that may be in liquid, solid and/or dryersheet form; as well as cleaning auxiliaries such as bleach additives and“stain-stick” or pre-treat types, substrate-laden compositions such asdryer added sheets. All of such compositions which are applicable may bein standard, concentrated or even highly concentrated form even to theextent that such compositions may in certain aspect be non-aqueous.

Method of Use

The present invention includes a method for cleaning any surfaceincluding treating a textile or a hard surface or other surfaces in thefield of fabric and/or home care. It is comtemplated that cleaning asdescribed may be both in small scale as in e.g. family house hold aswell as in large scale as in e.g. industrial and professional settings.In one aspect of the invention, the method comprises the step ofcontacting the surface to be treated in a pre-treatment step or mainwash step of a washing process, most preferably for use in a textilewashing step or alternatively for use in dishwashing including bothmanual as well as automated/mechanical dishwashing. In one embodiment ofthe invention the lipase variant and other components are addedsequentially into the method for cleaning and/or treating the surface.Alternatively, the lipase variant and other components are addedsimultaneously.

As used herein, washing includes but is not limited to, scrubbing, andmechanical agitation. Washing may be conducted with a foam compositionas described in WO08/101958 and/or by applying alternating pressure(pressure/vacuum) as an addition or as an alternative to scrubbing andmechanical agitation. Drying of such surfaces or fabrics may beaccomplished by any one of the common means employed either in domesticor industrial settings. The cleaning compositions of the presentinvention are ideally suited for use in laundry as well as dishwashingapplications. Accordingly, the present invention includes a method forcleaning an object including but not limiting to fabric, tableware,cutlery and kitchenware. The method comprises the steps of contactingthe object to be cleaned with a said cleaning composition comprising atleast one embodiment of Applicants' cleaning composition, cleaningadditive or mixture thereof. The fabric may comprise most any fabriccapable of being laundered in normal consumer or institutional useconditions. The solution may have a pH from 8 to 10.5. The compositionsmay be employed at concentrations from 500 to 15.000 ppm in solution.The water temperatures typically range from 5° C. to 90° C. The water tofabric ratio is typically from 1:1 to 30:1.

In one aspect the invention relates to a method of using the polypeptidewith at least 60% identity to SEQ ID NO: 2 for producing a composition.In one aspect the invention relates to use of the composition forcleaning an object.

In one aspect the invention relates to a method of producing thecomposition, comprising adding a polypeptide with at least 60% identityto SEQ ID NO: 2, and a surfactant. In one aspect the invention relatesto a method for cleaning a surface, comprising contacting a lipid stainpresent on the surface to be cleaned with the cleaning composition. Inone aspect the invention relates to a method for hydrolyzing a lipidpresent in a soil and/or a stain on a surface, comprising contacting thesoil and/or the stain with the cleaning composition. In one aspect theinvention relates to use of the composition in the hydrolysis of acarboxylic acid ester. In one aspect the invention relates to use of thecomposition in the hydrolysis, synthesis or interesterification of anester. In one aspect the invention relates to use of the composition forthe manufacture of a stable formulation.

Plants

The present invention also relates to plants, e.g., a transgenic plant,plant part, or plant cell, comprising a polynucleotide of the presentinvention so as to express and produce the variant in recoverablequantities. The variant may be recovered from the plant or plant part.Alternatively, the plant or plant part containing the variant may beused as such for improving the quality of a food or feed, e.g.,improving nutritional value, palatability, and rheological properties,or to destroy an antinutritive factor.

The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous(a monocot). Examples of monocot plants are grasses, such as meadowgrass (blue grass, Poa), forage grass such as Festuca, Lolium, temperategrass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley,rice, sorghum, and maize (corn).

Examples of dicot plants are tobacco, legumes, such as lupins, potato,sugar beet, pea, bean and soybean, and cruciferous plants (familyBrassicaceae), such as cauliflower, rape seed, and the closely relatedmodel organism Arabidopsis thaliana.

Examples of plant parts are stem, callus, leaves, root, fruits, seeds,and tubers as well as the individual tissues comprising these parts,e.g., epidermis, mesophyll, parenchyme, vascular tissues, meristems.Specific plant cell compartments, such as chloroplasts, apoplasts,mitochondria, vacuoles, peroxisomes and cytoplasm are also considered tobe a plant part. Furthermore, any plant cell, whatever the tissueorigin, is considered to be a plant part. Likewise, plant parts such asspecific tissues and cells isolated to facilitate the utilization of theinvention are also considered plant parts, e.g., embryos, endosperms,aleurone and seed coats.

Also included within the scope of the present invention are the progenyof such plants, plant parts, and plant cells.

The transgenic plant or plant cell expressing a variant may beconstructed in accordance with methods known in the art. In short, theplant or plant cell is constructed by incorporating one or moreexpression constructs encoding a variant into the plant host genome orchloroplast genome and propagating the resulting modified plant or plantcell into a transgenic plant or plant cell.

The expression construct is conveniently a nucleic acid construct thatcomprises a polynucleotide encoding a variant operably linked withappropriate regulatory sequences required for expression of thepolynucleotide in the plant or plant part of choice. Furthermore, theexpression construct may comprise a selectable marker useful foridentifying plant cells into which the expression construct has beenintegrated and DNA sequences necessary for introduction of the constructinto the plant in question (the latter depends on the DNA introductionmethod to be used).

The choice of regulatory sequences, such as promoter and terminatorsequences and optionally signal or transit sequences, is determined, forexample, on the basis of when, where, and how the variant is desired tobe expressed. For instance, the expression of the gene encoding avariant may be constitutive or inducible, or may be developmental, stageor tissue specific, and the gene product may be targeted to a specifictissue or plant part such as seeds or leaves. Regulatory sequences are,for example, described by Tague et al., 1988, Plant Physiology 86: 506.

For constitutive expression, the 35S-CaMV, the maize ubiquitin 1, or therice actin 1 promoter may be used (Franck et al., 1980, Cell 21:285-294; Christensen et al., 1992, Plant Mol. Biol. 18: 675-689; Zhanget al., 1991, Plant Cell 3: 1155-1165). Organ-specific promoters may be,for example, a promoter from storage sink tissues such as seeds, potatotubers, and fruits (Edwards and Coruzzi, 1990, Ann. Rev. Genet. 24:275-303), or from metabolic sink tissues such as meristems (Ito et al.,1994, Plant Mol. Biol. 24: 863-878), a seed specific promoter such asthe glutelin, prolamin, globulin, or albumin promoter from rice (Wu etal., 1998, Plant Cell Physiol. 39: 885-889), a Vicia faba promoter fromthe legumin B4 and the unknown seed protein gene from Vicia faba (Conradet al., 1998, J. Plant Physiol. 152: 708-711), a promoter from a seedoil body protein (Chen et al., 1998, Plant Cell Physiol. 39: 935-941),the storage protein napA promoter from Brassica napus, or any other seedspecific promoter known in the art, e.g., as described in WO 91/14772.Furthermore, the promoter may be a leaf specific promoter such as therbcs promoter from rice or tomato (Kyozuka et al., 1993, Plant Physiol.102: 991-1000), the chlorella virus adenine methyltransferase genepromoter (Mitra and Higgins, 1994, Plant Mol. Biol. 26: 85-93), the aldPgene promoter from rice (Kagaya et al., 1995, Mol. Gen. Genet. 248:668-674), or a wound inducible promoter such as the potato pin2 promoter(Xu et al., 1993, Plant Mol. Biol. 22: 573-588). Likewise, the promotermay be induced by abiotic treatments such as temperature, drought, oralterations in salinity or induced by exogenously applied substancesthat activate the promoter, e.g., ethanol, oestrogens, plant hormonessuch as ethylene, abscisic acid, and gibberellic acid, and heavy metals.

A promoter enhancer element may also be used to achieve higherexpression of a variant in the plant. For instance, the promoterenhancer element may be an intron that is placed between the promoterand the polynucleotide encoding a variant. For instance, Xu et al.,1993, supra, disclose the use of the first intron of the rice actin 1gene to enhance expression.

The selectable marker gene and any other parts of the expressionconstruct may be chosen from those available in the art.

The nucleic acid construct is incorporated into the plant genomeaccording to conventional techniques known in the art, includingAgrobacterium-mediated transformation, virus-mediated transformation,microinjection, particle bombardment, biolistic transformation, andelectroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990,Bio/Technology 8: 535; Shimamoto et al., 1989, Nature 338: 274).

Agrobacterium tumefaciens-mediated gene transfer is a method forgenerating transgenic dicots (for a review, see Hooykas andSchilperoort, 1992, Plant Mol. Biol. 19: 15-38) and for transformingmonocots, although other transformation methods may be used for theseplants. A method for generating transgenic monocots is particlebombardment (microscopic gold or tungsten particles coated with thetransforming DNA) of embryonic calli or developing embryos (Christou,1992, Plant J. 2: 275-281; Shimamoto, 1994, Curr. Opin. Biotechnol. 5:158-162; Vasil et al., 1992, Bio/Technology 10: 667-674). An alternativemethod for transformation of monocots is based on protoplasttransformation as described by Omirulleh et al., 1993, Plant Mol. Biol.21: 415-428. Additional transformation methods include those describedin U.S. Pat. Nos. 6,395,966 and 7,151,204 (both of which are hereinincorporated by reference in their entirety).

Following transformation, the transformants having incorporated theexpression construct are selected and regenerated into whole plantsaccording to methods well known in the art. Often the transformationprocedure is designed for the selective elimination of selection geneseither during regeneration or in the following generations by using, forexample, co-transformation with two separate T-DNA constructs or sitespecific excision of the selection gene by a specific recombinase.

In addition to direct transformation of a particular plant genotype witha construct of the present invention, transgenic plants may be made bycrossing a plant having the construct to a second plant lacking theconstruct. For example, a construct encoding a variant can be introducedinto a particular plant variety by crossing, without the need for everdirectly transforming a plant of that given variety. Therefore, thepresent invention encompasses not only a plant directly regenerated fromcells which have been transformed in accordance with the presentinvention, but also the progeny of such plants. As used herein, progenymay refer to the offspring of any generation of a parent plant preparedin accordance with the present invention. Such progeny may include a DNAconstruct prepared in accordance with the present invention. Crossingresults in the introduction of a transgene into a plant line by crosspollinating a starting line with a donor plant line. Non-limitingexamples of such steps are described in U.S. Pat. No. 7,151,204.

Plants may be generated through a process of backcross conversion. Forexample, plants include plants referred to as a backcross convertedgenotype, line, inbred, or hybrid.

Genetic markers may be used to assist in the introgression of one ormore transgenes of the invention from one genetic background intoanother. Marker assisted selection offers advantages relative toconventional breeding in that it can be used to avoid errors caused byphenotypic variations. Further, genetic markers may provide dataregarding the relative degree of elite germplasm in the individualprogeny of a particular cross. For example, when a plant with a desiredtrait which otherwise has a non-agronomically desirable geneticbackground is crossed to an elite parent, genetic markers may be used toselect progeny which not only possess the trait of interest, but alsohave a relatively large proportion of the desired germplasm. In thisway, the number of generations required to introgress one or more traitsinto a particular genetic background is minimized.

The present invention also relates to methods of producing a variant ofthe present invention comprising: (a) cultivating a transgenic plant ora plant cell comprising a polynucleotide encoding the variant underconditions conducive for production of the variant; and (b) recoveringthe variant.

Microcapsule Composition Comprising a Lipase Variant of the Invention

In this aspect, the invention relates to microcapsule compositions,wherein the membrane of the microcapsule is produced by cross-linking ofa polybranched polyamine having a molecular weight of more than 1 kDa,wherein the microcapsule comprising a lipase variant of the invention.

The membrane formed by crosslinking the polybranched polyamine iscapable of separating the enzyme from (anionic) surfactants in thedetergent, which are known to be detrimental to the stability ofenzymes.

A critically important parameter when using encapsulated enzymes indetergents is the ability to release the enzyme immediately upondilution of the detergent in water, as for example in a laundry ordishwash application. The microcapsules of the present invention haveexcellent properties in this regard, and are capable of releasing theentire encapsulated enzyme within a minute.

The microcapsules do not require the presence of a core polymer to becapable of releasing the enzyme, upon dilution in water. Further, theinvention does not require the enzyme(s) to be in a precipitated form inthe core of the microcapsule, in order to control premature release, asdescribed in WO 97/24177.

When encapsulating a lipase variant of the invention, and optionallyother enzymes, in a microcapsule with a semipermeable membrane, andhaving a water activity inside these capsules (prior to addition to theliquid detergent) higher than in the liquid detergent, the capsules willundergo a (partly) collapse when added to detergent (water is oozingout), thus leaving a more concentrated and more viscous enzymecontaining interior in the capsules. The collapse of the membrane mayalso result in a reduced permeability. This can be further utilized byaddition of stabilizers/polymers, especially ones that are not permeablethrough the membrane. The collapse and resulting increase in viscositywill reduce/hinder the diffusion of hostile components (e.g.,surfactants or sequestrants) into the capsules, and thus increase thestorage stability of the enzyme in liquid detergent. Components inliquid detergent that are sensitive to the enzyme (e.g., components thatact as substrate for the enzyme) are also protected against degradationby the enzyme. During wash the liquid detergent is diluted by water,thus increasing the water activity. Water will now diffuse into thecapsules (osmosis). The capsules will swell and the membrane will eitherbecome permeable to the enzyme so they can leave the capsules, or simplyburst and in this way releasing the enzyme. The concept is veryefficient in stabilizing the enzymes against hostile components inliquid detergent, and vice versa also protects enzyme sensitivecomponents in the liquid detergent from enzymes.

Examples of detergent components which are sensitive to, and can bedegraded by, enzymes include (relevant enzyme in parenthesis): xanthangum (xanthanase), polymers with ester bonds (lipase), hydrogenatedcastor oil (lipase), perfume (lipase), methyl ester sulfonatesurfactants (lipase), cellulose and cellulose derivatives (e.g. CMC)(cellulase), and dextrin and cyclodextrin (amylase).

Also, sensitive detergent ingredients can be encapsulated, and thusstabilized, in the microcapsules of the invention. Sensitive detergentingredients are prone to degradation during storage. Such detergentingredients include bleaching compounds, bleach activators, perfumes,polymers, builder, surfactants, etc.

Generally, the microcapsules of the invention can be used to separateincompatible components/compounds in detergents.

Addition of the microcapsules to detergents can be used to influence thevisual appearance of the detergent product, such as an opacifying effect(small microcapsules) or an effect of distinctly visible particles(large microcapsules). The microcapsules may also be colored.

The microcapsules can be used to reduce the enzyme dust levels duringhandling and processing of enzyme products.

Unless otherwise indicated, all percentages are indicated as percent byweight (% w/w) throughout the application.

Microcapsule

The microcapsules are typically produced by forming water droplets intoa continuum that is non-miscible with water—i.e., typically by preparinga water-in-oil emulsion—and subsequently formation of the membrane byinterfacial polymerization via addition of a cross-linking agent. Aftereventual curing the capsules can be harvested and further rinsed andformulated by methods known in the art. The capsule formulation issubsequently added to the detergent.

The payload, the major membrane constituents and eventual additionalcomponent that are to be encapsulated are found in the water phase. Inthe continuum is found components that stabilize the water dropletstowards coalescence (emulsifiers, emulsion stabilizers, surfactantsetc.) and the cross-linking agent is also added via the continuum.

The emulsion can be prepared be any methods known in the art, e.g., bymechanical agitation, dripping processes, membrane emulsification,microfluidics, sonication etc. In some cases simple mixing of the phasesautomatically will result in an emulsion, often referred to asself-emulsification. Using methods resulting in a narrow sizedistribution is an advantage.

The cross-linking agent(s) is typically subsequently added to theemulsion, either directly or more typically by preparing a solution ofthe crosslinking agent in a solvent which is soluble in the continuousphase. The emulsion and cross-linking agent or solution hereof can bemixed by conventional methods used in the art, e.g., by simple mixing orby carefully controlling the flows of the emulsion and the cross-linkingagent solution through an in-line mixer.

In some cases curing of the capsules is needed to complete the membraneformation. Curing is often simple stirring of the capsules for some timeto allow the interfacial polymerization reaction to end. In other casesthe membrane formation can be stopped by addition of reaction quencher.

The capsules may be post modified, e.g., by reacting components onto themembrane to hinder or reduce flocculation of the particles in thedetergent as described in WO 99/01534.

The produced capsules can be isolated or concentrated by methods knownin the art, e.g., by filtration, centrifugation, distillation ordecantation of the capsule dispersion.

The resulting capsules can be further formulated, e.g., by addition ofsurfactants to give the product the desired properties for storage,transport and later handling and addition to the detergent. Othermicrocapsule formulation agents include rheology modifiers, biocides(e.g., Proxel), acid/base for adjustment of pH (which will also adjustinside the microcapsules), and water for adjustment of water activity.

The capsule forming process may include the following steps:

-   Preparation of the initial water and oil phase(s),-   Forming a water-in-oil emulsion,-   Membrane formation by interfacial polymerization,-   Optional post modification,-   Optional isolation and/or formulation,-   Addition to detergent.

The process can be either a batch process or a continuous orsemi-continuous process.

A microcapsule according to the invention is a small aqueous sphere witha uniform membrane around it. The material inside the microcapsule isreferred to as the core, internal phase, or fill, whereas the membraneis sometimes called a shell, coating, or wall. The microcapsules of theinvention have diameters between 0.5 μm and 2 millimeters. Preferably,the mean diameter of the microcapsules is in the range of 1 μm to 1000μm, more preferably in the range of 5 μm to 500 μm, even more preferablyin the range of 10 μm to 500 μm, even more preferably in the range of 50μm to 500 μm, and most preferably in the range of 50 μm to 200 μm.Alternatively, the diameter of the microcapsules is in the range of 0.5μm to 30 μm; or in the range of 1 μm to 25 μm. The diameter of themicrocapsule is measured in the oil phase after polymerization iscomplete. The diameter of the capsule may change depending on the wateractivity of the surrounding chemical environment.

Microencapsulation of enzymes, as used in the present invention, may becarried out by interfacial polymerization, wherein the two reactants ina polymerization reaction meet at an interface and react rapidly. Thebasis of this method is a reaction of a polyamine with an acidderivative, usually an acid halide, acting as a crosslinking agent. Thepolyamine is preferably substantially water-soluble (when in free baseform). Under the right conditions, thin flexible membranes form rapidlyat the interface. One way of carrying out the polymerization is to usean aqueous solution of the enzyme and the polyamine, which areemulsified with a non-aqueous solvent (and an emulsifier), and asolution containing the acid derivative is added. An alkaline agent maybe present in the enzyme solution to neutralize the acid formed duringthe reaction. Polymer (polyamide) membranes form instantly at theinterface of the emulsion droplets. The polymer membrane of themicrocapsule is typically of a cationic nature, and thus bind/complexwith compounds of an anionic nature.

The diameter of the microcapsules is determined by the size of theemulsion droplets, which is controlled, for example by the stirringrate.

Emulsion

An emulsion is a temporary or permanent dispersion of one liquid phasewithin a second liquid phase. The second liquid is generally referred toas the continuous phase. Surfactants are commonly used to aid in theformation and stabilization of emulsions. Not all surfactants areequally able to stabilize an emulsion. The type and amount of asurfactant needs to be selected for optimum emulsion utility especiallywith regard to preparation and physical stability of the emulsion, andstability during dilution and further processing. Physical stabilityrefers to maintaining an emulsion in a dispersion form. Processes suchas coalescence, aggregation, adsorption to container walls,sedimentation and creaming, are forms of physical instability, andshould be avoided. Examples of suitable surfactants are described in WO97/24177, page 19-21; and in WO 99/01534.

Emulsions can be further classified as either simple emulsions, whereinthe dispersed liquid phase is a simple homogeneous liquid, or a morecomplex emulsion, wherein the dispersed liquid phase is a heterogeneouscombination of liquid or solid phases, such as a double emulsion or amultiple-emulsion. For example, a water-in-oil double emulsion ormultiple emulsion may be formed wherein the water phase itself furthercontains an emulsified oil phase; this type of emulsion may be specifiedas an oil-in-water-in oil (o/w/o) emulsion. Alternatively, awater-in-oil emulsion may be formed wherein the water phase contains adispersed solid phase often referred to as a suspension-emulsion. Othermore complex emulsions can be described. Because of the inherentdifficulty in describing such systems, the term emulsion is used todescribe both simple and more complex emulsions without necessarilylimiting the form of the emulsion or the type and number of phasespresent

Polyamine

The rigidity/flexibility and permeability of the membrane is mainlyinfluenced by the choice of polyamine. The polyamine is according to theinvention a polybranched polyamine. Each branch, preferably ending witha primary amino group serves as a tethering point in the membranenetwork, thereby giving the favorable properties of the invention. Apolybranched polyamine is according to the present invention a polyaminehaving more than two branching points and more than two reactive aminogroups (capable of reacting with the crosslinking agent, i.e., primaryand secondary amino groups). The polybranched polyamine is used asstarting material when the emulsion is prepared—it is not formed in situfrom other starting materials. To obtain the attractive properties ofthe invention, the polybranched structure of the polyamine must bepresent as starting material.

There is a close relation between number of branching points and numberof primary amines, since primary amines will always be positioned at theend of a branch: A linear amine can only contain two primary amines. Foreach branching point hypothetically introduced in such a linear di-aminewill allow one or more primary amine(s) to be introduced at the end ofthe introduced branch(es). In this context we understand the primaryamino group as part of the branch, i.e., the endpoint of the branch. Forexample, we consider both tris(2-aminoethyl)amine and1,2,3-propanetriamine as molecules having one branching point. For theinvention the polyamine has at least four primary amines. Branchingpoints can be introduced from an aliphatic hydrocarbon chain as in thepreviously stated examples or from unsaturated carbon bonds, such as in,e.g., 3,3′-diaminobenzidine, or from tertiary amino groups, such as inN,N,N′,N′-tetrakis-(2-aminoethyl)ethylenediamine.

In addition to the number of branching points, we have found that thecompactness of the reactive amino groups is of high importance. Asubstance such as, e.g.,N,N,N′,N′-tetrakis-(12-aminododecyl)ethylenediamine would not besuitable. Neither would a peptide or protein, such as an enzyme, besuitable for membrane formation. Thus, the polybranched polyamine is nota peptide or protein.

In an embodiment, the reactive amino groups constitute at least 15% ofthe molecular weight of the polybranched polyamine, such as more than20%, or more than 25%. Preferably, the molecular weight of thepolybranched polyamine is at least 1 kDa; more preferably, the molecularweight of the polybranched polyamine is at least 1.3 kDa.

In a preferred embodiment, the polybranched polyamine is apolyethyleneimine (PEI), and modifications thereof, having more than twobranching points and more than two reactive amino groups; wherein thereactive amino groups constitute at least 15% of the molecular weight ofthe PEI, such as more than 20%, or more than 25%. Preferably, themolecular weight of the PEI is at least 1 kDa.

Combinations of different polybranched polyamines may be used forpreparing the microcapsule according to the invention.

The advantageous properties (e.g., enzyme storage stability, reducedenzyme leakage, reduced in-flux of detergent ingredients) of themicrocapsule of the invention may be improved by adding one or moresmall amines with a molecular weight of less than 1 kDa. The small amineis preferably substantially water-soluble (when in free base form) andcan be a material such as ethylene diamine, hexamethylene diamine,hexane diamine, diethylene tetramine, ethylene tetramine, diaminobenzene, piperazine, tetramethylene pentamine or, preferably, diethylenetriamine (DETA). The small amines may be added in an amount of up to50%, preferably up to 40%, up to 30%, up to 20%, up to 10%, or up to 5%,by weight of the total content of small amine and polybranchedpolyamine, when preparing the microcapsule of the invention.

Crosslinking Agent

The crosslinking agent as used in the present invention is a moleculewith at least two groups/sites capable of reacting with amines to formcovalent bonds.

The crosslinking agent is preferably oil soluble and can be in the formof an acid anhydride or acid halide, preferably an acid chloride. Forexample, it can be adipoyl chloride, sebacoyl chloride, dodecanediocacid chloride, phthaloyl chloride, terephthaloyl chloride, isophthaloylchloride, or trimesoyl chloride; but preferably, the crosslinking agentis terephthaloyl chloride or trimesoyl chloride.

Enzyme(s)

In an embodiment, the composition or microcapsure composition of theinvention may further comprise an enzyme selected from the groupconsisting of protease, amylase, lipase, cellulase, mannanase,pectinase, DNAse, laccase, peroxidase, haloperoxidase, perhydrolase, andcombinations thereof.

The enzyme(s) in the composition or encapsulated in the microcapsule ofthe invention may include one or more enzymes suitable for including inlaundry or dishwash detergents (detergent enzymes) such as a protease(e.g., subtilisin or metalloprotease), lipase, cutinase, amylase,carbohydrase, cellulase, pectinase, mannanase, arabinase, galactanase,xanthanase, xylanase, DNAse, perhydrolase, oxidoreductase (e.g.,laccase, peroxidase, peroxygenase and/or haloperoxidase). Preferreddetergent enzymes are protease (e.g., subtilisin or metalloprotease),lipase, amylase, lyase, cellulase, pectinase, mannanase, DNAse,perhydrolase, and oxidoreductases (e.g., laccase, peroxidase,peroxygenase and/or haloperoxidase); or combinations thereof. Morepreferred detergent enzymes are protease (e.g., subtilisin ormetalloprotease), lipase, amylase, cellulase, pectinase, and mannanase;or combinations thereof.

The composition or microcapsule composition of the invention may includemore than 0.1% (w/w) active enzyme protein, in particular lipase variantof the invention; preferably more than 0.25%, more preferably more than0.5%, more preferably more than 1%, more preferably more than 2.5%, morepreferably more than 5%, more preferably more than 7.5%, more preferablymore than 10%, more preferably more than 12.5%, more preferably morethan 15%, even more preferably more than 20%, and most preferably morethan 25% (w/w) active enzyme protein.

Proteases: The proteases for use in the present invention are serineproteases, such as subtilisins, metalloproteases and/or trypsin-likeproteases. Preferably, the proteases are subtilisins ormetalloproteases; more preferably, the proteases are subtilisins.

A serine protease is an enzyme which catalyzes the hydrolysis of peptidebonds, and in which there is an essential serine residue at the activesite (White, Handler and Smith, 1973 “Principles of Biochemistry,” FifthEdition, McGraw-Hill Book Company, NY, pp. 271-272). Subtilisinsinclude, preferably consist of, the I-S1 and I-S2 sub-groups as definedby Siezen et al., Protein Engng. 4 (1991) 719-737; and Siezen et al.,Protein Science 6 (1997) 501-523. Because of the highly conservedstructure of the active site of serine proteases, the subtilisinaccording to the invention may be functionally equivalent to theproposed sub-group designated subtilase by Siezen et al. (supra).

The subtilisin may be of animal, vegetable or microbial origin,including chemically or genetically modified mutants (protein engineeredvariants), preferably an alkaline microbial subtilisin. Examples ofsubtilisins are those derived from Bacillus, e.g., subtilisin Novo,subtilisin Carlsberg, subtilisin BPN′, subtilisin 309, subtilisin 147and subtilisin 168 (described in WO 89/06279) and Protease PD138 (WO93/18140). Examples are described in WO 98/020115, WO 01/44452, WO01/58275, WO 01/58276, WO 03/006602 and WO 04/099401. Examples oftrypsin-like pro¬teases are trypsin (e.g., of por¬cine or bovine origin)and the Fusarium protease described in WO 89/06270 and WO 94/25583.Other examples are the variants described in WO 92/19729, WO 88/08028,WO 98/20115, WO 98/20116, WO 98/34946, WO 2000/037599, WO 2011/036263,especially the variants with substitutions in one or more of thefollowing positions: 27, 36, 57, 76, 87, 97, 101, 104, 120, 123, 167,170, 194, 206, 218, 222, 224, 235, and 274.

The metalloprotease may be of animal, vegetable or microbial origin,including chemically or genetically modified mutants (protein engineeredvariants), preferably an alkaline microbial metalloprotease. Examplesare described in WO 2007/044993, WO 2012/110562 and WO 2008/134343.

Examples of commercially available subtilisins include Kannase™,Everlase™, Relase™, Esperase™, Alcalase™, Durazym™, Savinase™, Ovozyme™,Liquanase™, Coronase™, Polarzyme™, Pyrase™, Pancreatic Trypsin NOVO(PTN), Bio-Feed™ Pro and Clear-Lens™ Pro; Blaze (all available fromNovozymes NS, Bagsvaerd, Denmark). Other commercially availableproteases include Neutrase™, Ronozyme™ Pro, Maxatase™, Maxacal™,Maxapem™, Opticlean™, Properase™, Purafast™, Purafect™, Purafect Ox™,Purafact Prime™, Excellase™, FN2™, FN3™ and FN4™ (available fromNovozymes, Genencor International Inc., Gist-Brocades, BASF, or DSM).Other examples are Primase™ and Duralase™. Blap R, Blap S and Blap Xavailable from Henkel are also examples.

Lyases: The lyase may be a pectate lyase derived from Bacillus,particularly B. licheniformis or B. agaradhaerens, or a variant derivedof any of these, e.g. as described in U.S. Pat. No. 6,124,127, WO99/027083, WO 99/027084, WO 02/006442, WO 02/092741, WO 03/095638,Commercially available pectate lyases are XPect; Pectawash and Pectaway(Novozymes NS).

Mannanase: The mannanase may be an alkaline mannanase of Family 5 or 26.It may be a wild-type from Bacillus or Humicola, particularly B.agaradhaerens, B. licheniformis, B. halodurans, B. clausii, or H.insolens. Suitable mannanases are described in WO 99/064619. Acommercially available mannanase is Mannaway (Novozymes NS).

Cellulases: Suitable cellulases include those of bacterial or fungalorigin. Chemically modified or protein engineered mutants are included.Suitable cellulases include cellulases from the genera Bacillus,Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, e.g., the fungalcellulases produced from Humicola insolens, Myceliophthora thermophilaand Fusarium oxysporum disclosed in U.S. Pat. Nos. 4,435,307, 5,648,263,5,691,178, 5,776,757 and WO 89/09259.

Especially suitable cellulases are the alkaline or neutral cellulaseshaving color care benefits. Examples of such cellulases are cellulasesdescribed in EP 0 495 257, EP 0 531 372, WO 96/11262, WO 96/29397, WO98/08940. Other examples are cellulase variants such as those describedin WO 94/07998, EP 0 531 315, U.S. Pat. Nos. 5,457,046, 5,686,593,5,763,254, WO 95/24471, WO 98/12307 and PCT/DK98/00299.

Commercially available cellulases include Celluzyme™, and Carezyme™(Novozymes NS), Clazinase™, and Puradax HA™ (Genencor InternationalInc.), and KAC-500(B)™ (Kao Corporation).

Besides a lipase variant of the invention the composition ormicrocapsule composition may comprise other lipases.

Other Lipases and Cutinases: Suitable lipases and cutinases includethose of bacterial or fungal origin. Chemically modified or proteinengineered mutants are included. Examples include lipase fromThermomyces, e.g., from T. lanuginosus (previously named Humicolalanuginosa) as described in EP 258 068 and EP 305 216, cutinase fromHumicola, e.g., H. insolens as described in WO 96/13580, a Pseudomonaslipase, e.g., from P. alcaligenes or P. pseudoalcaligenes (EP 218 272),P. cepacia (EP 331 376), P. stutzeri (GB 1,372,034), P. fluorescens,Pseudomonas sp. strain SD 705 (WO 95/06720 and WO 96/27002), P.wisconsinensis (WO 96/12012), a Bacillus lipase, e.g., from B. subtilis(Dartois et al., 1993, Biochemica et Biophysica Acta, 1131: 253-360), B.stearothermophilus (JP 64/744992) or B. pumilus (WO 91/16422).

Other examples are lipase variants such as those described in WO92/05249, WO 94/01541, EP 407 225, EP 260 105, WO 95/35381, WO 96/00292,WO 95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO 97/04079, WO97/07202, WO 00/060063, WO 2007/087508 and WO 2009/109500.

Other commercially available lipase enzymes include Lipolase™, LipolaseUltra™, and Lipex™; Lipex Evity 100L, Lecitase™, Lipolex™; Lipoclean™,Lipoprime™ (Novozymes NS). Other commercially available lipases includeLumafast (Genencor Int Inc); Lipomax (Gist-Brocades/Genencor Int Inc)and Bacillus sp. lipase from Solvay.

Amylases: Suitable amylases include those of bacterial or fungal origin.Chemically modified or protein engineered mutants are included. Amylasesinclude, for example, alpha-amylases obtained from Bacillus, e.g., aspecial strain of Bacillus licheniformis, described in more detail in GB1,296,839.

Examples of suitable amylases include amylases having SEQ ID NO: 2 in WO95/10603 or variants having 90% sequence identity to SEQ ID NO: 3thereof. Preferred variants are described in WO 94/02597, WO 94/18314,WO 97/43424 and SEQ ID NO: 4 of WO 99/019467, such as variants withsubstitutions in one or more of the following positions: 15, 23, 105,106, 124, 128, 133, 154, 156, 178, 179, 181, 188, 190, 197, 201, 202,207, 208, 209, 211, 243, 264, 304, 305, 391, 408, and 444.

Different suitable amylases include amylases having SEQ ID NO: 6 in WO02/010355 or variants thereof having 90% sequence identity to SEQ ID NO:6. Preferred variants of SEQ ID NO: 6 are those having a deletion inpositions 181 and 182 and a substitution in position 193. Other amylaseswhich are suitable are hybrid alpha-amylase comprising residues 1-33 ofthe alpha-amylase derived from B. amyloliquefaciens shown in SEQ ID NO:6 of WO 2006/066594 and residues 36-483 of the B. licheniformisalpha-amylase shown in SEQ ID NO: 4 of WO 2006/066594 or variants having90% sequence identity thereof. Preferred variants of this hybridalpha-amylase are those having a substitution, a deletion or aninsertion in one of more of the following positions: G48, T49, G107,H156, A181, N190, M197, I201, A209 and Q264. Most preferred variants ofthe hybrid alpha-amylase comprising residues 1-33 of the alpha-amylasederived from B. amyloliquefaciens shown in SEQ ID NO: 6 of WO2006/066594 and residues 36-483 of SEQ ID NO: 4 are those having thesubstitutions:

-   M197T;-   H156Y+A181T+N190F+A209V+Q264S; or-   G48A+T49I+G107A+H156Y+A181T+N190F+I201F+A209V+Q2645.

Further amylases which are suitable are amylases having SEQ ID NO: 6 inWO 99/019467 or variants thereof having 90% sequence identity to SEQ IDNO: 6. Preferred variants of SEQ ID NO: 6 are those having asubstitution, a deletion or an insertion in one or more of the followingpositions: R181, G182, H183, G184, N195, I206, E212, E216 and K269.Particularly preferred amylases are those having deletion in positionsR181 and G182, or positions H183 and G184.

Additional amylases which can be used are those having SEQ ID NO: 1, SEQID NO: 3, SEQ ID NO: 2 or SEQ ID NO: 7 of WO 96/023873 or variantsthereof having 90% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQID NO: 3 or SEQ ID NO: 7. Preferred variants of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3 or SEQ ID NO: 7 are those having a substitution, adeletion or an insertion in one or more of the following positions: 140,181, 182, 183, 184, 195, 206, 212, 243, 260, 269, 304 and 476. Morepreferred variants are those having a deletion in positions 181 and 182or positions 183 and 184. Most preferred amylase variants of SEQ ID NO:1, SEQ ID NO: 2 or SEQ ID NO: 7 are those having a deletion in positions183 and 184 and a substitution in one or more of positions 140, 195,206, 243, 260, 304 and 476.

Other amylases which can be used are amylases having SEQ ID NO: 2 of WO08/153815, SEQ ID NO: 10 in WO 01/66712 or variants thereof having 90%sequence identity to SEQ ID NO: 2 of WO 08/153815 or 90% sequenceidentity to SEQ ID NO: 10 in WO 01/66712. Preferred variants of SEQ IDNO: 10 in WO 01/66712 are those having a substitution, a deletion or aninsertion in one of more of the following positions: 176, 177, 178, 179,190, 201, 207, 211 and 264.

Further suitable amylases are amylases having SEQ ID NO: 2 of WO09/061380 or variants having 90% sequence identity to SEQ ID NO: 2thereof. Preferred variants of SEQ ID NO: 2 are those having atruncation of the C-terminus and/or a substitution, a deletion or aninsertion in one of more of the following positions: Q87, Q98, S125,N128, T131, T165, K178, R180, S181, T182, G183, M201, F202, N225, S243,N272, N282, Y305, R309, D319, Q320, Q359, K444 and G475. More preferredvariants of SEQ ID NO: 2 are those having the substitution in one ofmore of the following positions: Q87E,R, Q98R, S125A, N128C, T131I,T1651, K178L, T182G, M201L, F202Y, N225E,R, N272E,R, S243Q,A,E,D, Y305R,R309A, Q320R, Q359E, K444E and G475K and/or deletion in position R180and/or S181 or of T182 and/or G183. Most preferred amylase variants ofSEQ ID NO: 2 are those having the substitutions:

-   N128C+K178L+T182G+Y305R+G475K;-   N128C+K178L+T182G+F202Y+Y305R+D319T+G475K;-   S125A+N128C+K178L+T182G+Y305R+G475K; or-   S125A+N128C+T131I+T165I+K178L+T182G+Y305R+G475K wherein the variants    are C-terminally truncated and optionally further comprises a    substitution at position 243 and/or a deletion at position 180    and/or position 181.

Other suitable amylases are the alpha-amylase having SEQ ID NO: 12 inWO01/66712 or a variant having at least 90% sequence identity to SEQ IDNO: 12. Preferred amylase variants are those having a substitution, adeletion or an insertion in one of more of the following positions ofSEQ ID NO: 12 in WO01/66712: R28, R118, N174; R181, G182, D183, G184,G186, W189, N195, M202, Y298, N299, K302, S303, N306, R310, N314; R320,H324, E345, Y396, R400, W439, R444, N445, K446, Q449, R458, N471, N484.Particular preferred amylases include variants having a deletion of D183and G184 and having the substitutions R118K, N195F, R320K and R458K, anda variant additionally having substitutions in one or more positionselected from the group: M9, G149, G182, G186, M202, T257, Y295, N299,M323, E345 and A339, most preferred a variant that additionally hassubstitutions in all these positions.

Other examples are amylase variants such as those described inWO2011/098531, WO2013/001078 and WO2013/001087.

Commercially available amylases are Stainzyme; Stainzyme Plus; Duramyl™,Termamyl™, Termamyl Ultra; Natalase, Fungamyl™ and BAN™ (Novozymes NS),Rapidase™ and Purastar™/Effectenz™, Powerase and Preferenz S100 (fromGenencor International Inc./DuPont).

Deoxyribonuclease (DNase): Suitable deoxyribonucleases (DNases) are anyenzyme that catalyzes the hydrolytic cleavage of phosphodiester linkagesin the DNA backbone, thus degrading DNA. According to the invention, aDNase which is obtainable from a bacterium is preferred; in particular aDNase which is obtainable from a Bacillus is preferred; in particular aDNase which is obtainable from Bacillus subtilis or Bacilluslicheniformis is preferred. Examples of such DNases are described inpatent application WO 2011/098579 or in PCT/EP2013/075922.

Perhydrolases: Suitable perhydrolases are capable of catalyzing aperhydrolysis reaction that results in the production of a peracid froma carboxylic acid ester (acyl) substrate in the presence of a source ofperoxygen (e.g., hydrogen peroxide). While many enzymes perform thisreaction at low levels, perhydrolases exhibit a highperhydrolysis:hydrolysis ratio, often greater than 1. Suitableperhydrolases may be of plant, bacterial or fungal origin. Chemicallymodified or protein engineered mutants are included.

Examples of useful perhydrolases include naturally occurringMycobacterium perhydrolase enzymes, or variants thereof. An exemplaryenzyme is derived from Mycobacterium smegmatis. Such enzyme, itsenzymatic properties, its structure, and variants thereof, are describedin WO 2005/056782, WO 2008/063400, US 2008/145353, and US2007167344.

Oxidases/peroxidases: Suitable oxidases and peroxidases (oroxidoreductases) include various sugar oxidases, laccases, peroxidasesand haloperoxidases.

Suitable peroxidases include those comprised by the enzymeclassification EC 1.11.1.7, as set out by the Nomenclature Committee ofthe International Union of Biochemistry and Molecular Biology (IUBMB),or any fragment derived therefrom, exhibiting peroxidase activity.

Suitable peroxidases include those of plant, bacterial or fungal origin.Chemically modified or protein engineered mutants are included. Examplesof useful peroxidases include peroxidases from Coprinopsis, e.g., fromC. cinerea (EP 179,486), and variants thereof as those described in WO93/24618, WO 95/10602, and WO 98/15257.

A peroxidase for use in the invention also include a haloperoxidaseenzyme, such as chloroperoxidase, bromoperoxidase and compoundsexhibiting chloroperoxidase or bromoperoxidase activity. Haloperoxidasesare classified according to their specificity for halide ions.Chloroperoxidases (E.C. 1.11.1.10) catalyze formation of hypochloritefrom chloride ions.

In an embodiment, the haloperoxidase is a chloroperoxidase. Preferably,the haloperoxidase is a vanadium haloperoxidase, i.e., avanadate-containing haloperoxidase. In a preferred method of the presentinvention the vanadate-containing haloperoxidase is combined with asource of chloride ion.

Haloperoxidases have been isolated from many different fungi, inparticular from the fungus group dematiaceous hyphomycetes, such asCaldariomyces, e.g., C. fumago, Alternaria, Curvularia, e.g., C.verruculosa and C. inaequalis, Drechslera, Ulocladium and Botrytis.

Haloperoxidases have also been isolated from bacteria such asPseudomonas, e.g., P. pyrrocinia and Streptomyces, e.g., S.aureofaciens.

In an preferred embodiment, the haloperoxidase is derivable fromCurvularia sp., in particular Curvularia verruculosa or Curvulariainaequalis, such as C. inaequalis CBS 102.42 as described in WO95/27046; or C. verruculosa CBS 147.63 or C. verruculosa CBS 444.70 asdescribed in WO 97/04102; or from Drechslera hartlebii as described inWO 01/79459, Dendryphiella salina as described in WO 01/79458,Phaeotrichoconis crotalarie as described in WO 01/79461, orGeniculosporium sp. as described in WO 01/79460.

An oxidase according to the invention include, in particular, anylaccase enzyme comprised by the enzyme classification EC 1.10.3.2, orany fragment derived therefrom exhibiting laccase activity, or acompound exhibiting a similar activity, such as a catechol oxidase (EC1.10.3.1), an o-aminophenol oxidase (EC 1.10.3.4), ora bilirubin oxidase(EC 1.3.3.5).

Preferred laccase enzymes are enzymes of microbial origin. The enzymesmay be derived from plants, bacteria or fungi (including filamentousfungi and yeasts).

Suitable examples from fungi include a laccase derivable from a strainof Aspergillus, Neurospora, e.g., N. crassa, Podospora, Botrytis,Collybia, Fomes, Lentinus, Pleurotus, Trametes, e.g., T. villosa and T.versicolor, Rhizoctonia, e.g., R. solani, Coprinopsis, e.g., C. cinerea,C. comatus, C. friesii, and C. plicatilis, Psathyrella, e.g., P.condelleana, Panaeolus, e.g., P. papilionaceus, Myceliophthora, e.g., M.thermophila, Schytalidium, e.g., S. thermophilum, Polyporus, e.g., P.pinsitus, Phlebia, e.g., P. radiata (WO 92/01046), or Coriolus, e.g., C.hirsutus (JP 2238885).

Suitable examples from bacteria include a laccase derivable from astrain of Bacillus.

A laccase derived from Coprinopsis or Myceliophthora is preferred; inparticular a laccase derived from Coprinopsis cinerea, as disclosed inWO 97/08325; or from Myceliophthora thermophila, as disclosed in WO95/33836.

Examples of other oxidases include, but are not limited to, amino acidoxidase, glucose oxidase, lactate oxidase, galactose oxidase, polyoloxidase (e.g., WO2008/051491), and aldose oxidase. Oxidases and theircorresponding substrates may be used as hydrogen peroxide generatingenzyme systems, and thus a source of hydrogen peroxide. Several enzymes,such as peroxidases, haloperoxidases and perhydrolases, require a sourceof hydrogen peroxide. By studying EC 1.1.3._, EC 1.2.3._, EC 1.4.3._,and EC 1.5.3._ or similar classes (under the International Union ofBiochemistry), other examples of such combinations of oxidases andsubstrates are easily recognized by one skilled in the art.

Enzyme Stabilizers and/or Rheology Modifiers

The compositions or microcapsures may also contain enzyme stabilizers asknown in the art, e.g., polyols, polymers, reversible enzyme inhibitors,divalent cations, enzyme substrates, antioxidants etc. Water solublestabilizers are preferred.

Addition of slowly dissolving stabilizers can be used to create a localenvironment inside the capsule, which is more “friendly” to theencapsulated enzyme/compound, thus improving the stability duringstorage.

Examples of reversible protease inhibitors are boronic acids, peptidealdehydes and derivatives hereof and high molecular protein-typeinhibitors (like BASI/RASI inhibitors, see WO 2009/095425). An exampleof metalloprotease inhibitors is described in WO 2008/134343. Proteaseinhibitors are described in more detail below under the heading“Protease Inhibitors”.

Stabilizing polymers can be based on, e.g., polyvinylypyrrolidon,polyvinylacetate, polyvinylalcohol and copolymers hereof. Stabilizingpolyols can be smaller molecules like glycerol, sorbitol, propyleneglycol etc. but also larger molecules like polyethylene glycol,polysaccharides etc.

Of stabilizing divalent cations Ca2+, Mg2+ and Zn2+ are well-known inthe art. Thus, in an embodiment, the composition of the inventioncomprise a source of Ca2+, Mg2+ or Zn2+ ions. Preferably, the source ofCa2+, Mg2+ or Zn2+ ions is a poorly soluble (slowly dissolving) salt ofCa2+, Mg2+ or Zn2+. Poorly soluble means that the solubility in purewater at 20° C. is less than 5 g/l, 2 g/l, 1 g/l, 0.5 g/l, 0.2 g/l, 0.1g/l, or 0.05 g/l. Preferred salts of Ca2+, Mg2+ or Zn2+ are calciumcarbonate, magnesium carbonate, zinc carbonate, calcium sulfate, calciumsulfite, magnesium sulfite, zinc sulfite, calcium phosphate, dicalciumphosphate, magnesium phosphate, zinc phosphate, calcium citrate,magnesium citrate, zinc citrate, calcium oxalate, magnesium oxalate,zinc oxalate, calcium tartrate, magnesium tartrate, or zinc tartrate.

Also, slowly dissolving acids or bases can be used to create a local pHinside the microcapsule, which is more “friendly” to the encapsulatedenzyme/compound.

Enzymes are in most cases stabilized by addition of their substrates(e.g., protein for proteases, starch for amylases etc.). Antioxidants orreducing agents can be applied to reduce oxidation of enzymes, e.g.,thiosulfate, ascorbate etc. The net dosage needed of these stabilizersper gram detergent is much lower compared to adding the stabilizers tothe continuous detergent phase, as they are concentrated in the internalcapsule phase, and will in many cases either not diffuse out duringstorage, or only slowly diffuse out depending on the structure andmolecular weight of the stabilizer. Especially high molecular weightstabilizers (e.g., higher than 1 kDa, or higher than 2 kDa morepreferred higher than 5 kDa) will give improved net efficiency. Highmolecular weight inhibitors, polymers, polyols, cations, enzymesubstrates and antioxidants are thus preferred.

The enzyme may be protected by addition of a “scavenger” protein.Components destabilizing enzyme by reacting onto amino acid groups(e.g., amines) on the protein may thus react with the scavenger orsacrificial protein added. Scavenger protein with a sufficient largemolecular weight to stay inside the capsules are preferred.

A somewhat different way to improve the enzyme stability is to addrheology modifying components that increase viscosity of the internalcapsule phase. An increased internal viscosity will slow down diffusionof enzyme destabilizers into the capsules (and/or slow down thediffusion of enzyme stabilizers out of the capsule) and thus prolong thelifetime of the enzyme. Examples of such viscosity modifiers arepolymers like polyethylene glycol (PEG), polyethylene oxide (PEO),hydrophilic polyurethane, polyvinylpyrrolidon (PVP) and PVP vinylacetate copolymers, starch, hyaluronic acid, water soluble cellulosederivatives like carboxymethyl cellulose, water soluble gums like gumArabic, locust bean gum, guar gum or xanthan gum etc. and combinationsor copolymers hereof. Most preferred are nonionic high molecular weightpolymers, with a molecular weight higher than 1 kDa, or higher than 2kDa, more preferred higher than 5 kDa. Nonionic polymers are preferredas they in most cases are more compatible with the reactive membranepolymer than ionic polymers.

The high viscosity can either be accomplished by producing the capsulesusing a high viscosity aqueous phase, or—more sophisticated—producingcapsules where the viscosity increase first occur after producing theemulsion/capsules. This “triggered” viscosity increase is preferable aspreparing emulsions with a high viscosity aqueous phase can bedifficult. Triggered viscosity increase can be done in situ when addedto detergent by the internal capsule phase having a higher wateractivity than the detergent to which it is added, thus water willdiffuse out of the capsules (but not the rheology modifier) increasingthe viscosity of the internal phase after addition to detergent. Thiscan also be utilized using diffusion of salt or other low molecularcomponents, e.g., by having a component that will increase viscositywhen salt concentration is reduced by addition to detergent (e.g., apolymer that is precipitated at the initial high salt content butsoluble when salt concentration is reduced due to diffusion of salt whenadded to detergent). Another way to trigger viscosity is to usecomponents where the viscosity is dependent on the pH. For someinterfacial polymerization processes (e.g., amine—acid halogen reaction)the pH of the internal phase will change during encapsulation, in thecase of amine-acid halogen pH will be reduced during the interfacialpolymerization. This can be used to trigger an increase in viscosity.Many rheology modifiers like polyacrylates show a viscosity maximum at aspecific pH or pH range. Carbopol 934 from Lubrizol and Texipol 63-258from Scott-Bader are examples of rheology modifiers where viscosity issignificantly increased when reducing the pH from 11 to 8, or increasingpH from 4 to 8. Another polymer type with a different viscosity at lowpH and at high pH is partially hydrolyzed polyacrylamide. Yet anotherpossibility is to use rheology modifiers which are temperaturedependent, thus making the emulsion/encapsulation at one temperature,and subsequently changing the temperature to increase viscosity. Also alight or ultrasound induced viscosity can be utilized. Yet anothermethod is to use shear-thinning rheology modifiers, such that theviscosity is low at high shear when the emulsion is formed and high whenshear is reduced.

Another stabilization technique is to assure that the enzyme isprecipitated in the capsules during storage, for example by addition ofprecipitants like salt or polyethylene glycol (PEG). The same “triggeredstabilization” as described above can be used, e.g., by addition of PEG,which after addition to detergent is concentrated by water diffusing outto a degree where the enzyme will precipitate. In this way the enzymecan be in solution during processing of the capsules, but precipitatedwhen added to detergent.

Enzymes can also be used in precipitated or crystal form when preparingthe microcapsules.

In a specifically contemplated embodiment, the microcapsule compositionof the invention is one described in WO 2014/177709 (hereby incorporatedby reference) comprising a lipase variant of the invention.

In another embodiment, the invention relates to microcapsulecompositions, comprising a lipase variant of the invention entrapped ina compartment formed by a membrane, which membrane is produced bycross-linking of (a) a polybranched polyamine having a molecular weightof more than 800 Da, and (b) an aliphatic or aromatic amine having amolecular weight of less than 300 Da; wherein the weight ratio of(a)/(b) is in the range of 0.1 to 1000.

As mentioned above microcapsules may further comprising an enzymeselected from the group consisting of protease, metalloprotease,subtilisin, amylase, lipase, cutinase, cellulase, mannanase, pectinase,xanthanase, DNAse, laccase, peroxidase, haloperoxidase, perhydrolase,and combinations thereof. Other enzymes mentioned above are alsocontemplated.

In an embodiment, the reactive amino groups of the polybranchedpolyamine constitute at least 15% of the molecular weight. In anembodiment, the diameter of the compartment is at least 50 micrometers.In a preferred embodiment, the compartment contains at least 1% activeenzyme by weight of the total compartment, in particular lipase variantof the invention. As also mentioned above the microcapsules may furtherinclude an alcohol, such as a polyol.

In a preferred embodiment (a) is a polyethyleneimine.

In an embodiment (b) is an ethyleneamine or alkanolamine. In anpreferred embodiment (b) is selected from the group consisting ofethylene diamine, diethylene triamine, triethylene tetraamine,bis(3-aminopropyl)amine, monoethanolamine, diethanolamine,triethanolamine, hexamethylene diamine, diamino benzene, piperazine, andtetraethylene pentamine. In a more preferred embodiment (b) is selectedfrom the group consisting of diethylene triamine, triethylenetetraamine, bis(3-aminopropyl)amine, monoethanolamine, anddiethanolamine.

According to the invention the compartment of the microcapsule comprisesa source of Mg2+, Ca2+, or Zn2+ ions, such as a poorly soluble salt ofMg2+, Ca2+, or Zn2+.

In a preferred embodiment, the membrane is produced by using an acidchloride as crosslinking agent, such as isophtaloyl chloride,terephthaloyl chloride, or trimesoyl chloride. In a preferredembodiment, the membrane is produced by interfacial polymerization.

In a specifically contemplated embodiment, the microcapsule compositionof the invention is one described in WO 2015/1144784 (herebyincorporated by reference) comprising a lipase variant of the invention.

Liquid Products

In a final aspect, the invention relates to liquid products, comprisinga microcapsule composition of the invention. In a preferred embodiment,the liquid product comprises water or at least a significant amount ofwater.

The following paragraphs describe embodiments of the invention:

-   1. A variant of a parent lipase which variant has lipase activity,    has at least 60%, but less than 100% sequence identity with SEQ ID    NO: 2 and comprises one or more (e.g. several) substitutions at    positions corresponding to G23S, D27N, A40I, F51I,L, E56R, D57N,    V60E,K, K98I, N101D, R118F, G163S, Y220F, T231R, N233R, T244E, and    P256T.-   2. The variant of paragraph 1, wherein the variant comprises    substitutions at positions corresponding to T231R+N233R and one or    more (e.g., several) substitutions at positions corresponding to    G23S, D27N, A40I, F51I,L, E56R, D57N, V60E,K, K98I, N101D, R118F,    G163S, Y220F, T244E, and P256T.-   3. The variant of paragraph 1 or 2 comprising substitutions    corresponding to any of the following set of substitutions:

G23S + T231R + N233R D27N + T231R + N233R A40I + T231R + N233R F51I +T231R + N233R F51L + T231R + N233R E56R + T231R + N233R D57N + T231R +N233R V60E + T231R + N233R V60K + T231R + N233R K98I + T231R + N233RN101D + T231R + N233R R118F + T231R + N233R G163S + T231R + N233RY220F + T231R + N233R T231R + N233R + T244E T231R + N233R + P256T

-   4. The variant of any of paragraphs 1-3, wherein the variant    comprises substitutions corresponding to E56R+T231R+N233R and one or    more (e.g., several) substitutions at positions corresponding to    G23S, D27N, A40I, F51I,L, D57N, V60E,K, K98I, N101D, R118F, G163S,    Y220F, T244E, and P256T.-   5. The variant of any of paragraphs 1-4, wherein the variant    comprises substitutions at positions corresponding to    R118F+T231R+N233R and one or more (e.g., several) substitutions at    positions corresponding to G23S, D27N, A40I, F51I,L, E56R, D57N,    V60E,K, K98I, N101 D, G163S, Y220F, T244E, and P256T.-   6. The variant of any of paragraphs 1-5, wherein the variant    comprises substitutions at positions corresponding to    E56R+R118F+T231R+N233R and one or more (e.g., several) substitutions    at positions corresponding to G23S, D27N, A40I, F51I,L, D57N,    V60E,K, K98I, N101 D, G163S, Y220F, T244E, and P256T.-   7. The variant of any of paragraphs 1-6, wherein the variant    comprises substitutions at positions corresponding to    E56R+R118F+T231R+N233R+P256T and one or more (e.g., several)    substitutions at positions corresponding to G23S, D27N, A40I,    F51I,L, D57N, V60E,K, K98I, N101 D, G163S, Y220F, and T244E.-   8. The variant of any of paragraphs 1-7, wherein the variant    comprises substitutions at positions corresponding to    F51I,L+E56R+R118F+T231R+N233R+P256T and one or more (e.g., several)    substitutions at positions corresponding to G23S, D27N, A40I, D57N,    V60E,K, K98I, N101D, G163S, Y220F, and T244E.-   9. The variant of any of paragraphs 1-8, wherein the variant    comprises substitutions at positions corresponding to    G23S+F51I,L+E56R+R118F+T231R+N233R+P256T and one or more (e.g.,    several) substitutions at positions corresponding to D27N, A40I,    D57N, V60E,K, K98I, N101D, G163S, Y220F, and T244E.-   10. The variant of any of paragraphs 1-9, wherein the variant    comprises substitutions at positions corresponding to    D27N+F51I,L+E56R+R118F+T231R+N233R+P256T and one or more (e.g.,    several) substitutions at positions corresponding to G23S, A40I,    D57N, V60E,K, K98I, N101D, G163S, Y220F, and T244E.-   11. The variant of any of paragraphs 1-10, wherein the variant    comprises substitutions at positions corresponding to    A40I+F51I,L+E56R+R118F+T231R+N233R+P256T and one or more (e.g.,    several) substitutions at positions corresponding to G23S, D27N,    D57N, V60E,K, K98I, N101D, G163S, Y220F, and T244E.-   12. The variant of any of paragraphs 1-11, wherein the variant    comprises substitutions at positions corresponding to    F51I,L+E56R+D57N+R118F+T231R+N233R+P256T and one or more (e.g.,    several) substitutions at positions corresponding to G23S, D27N,    A40I, V60E,K, K98I, N101D, G163S, Y220F, and T244E.-   13. The variant of any of paragraphs 1-12, wherein the variant    comprises substitutions at positions corresponding to    F51I,L+E56R+D57N+K98I+R118F+T231R+N233R+P256T and one or more (e.g.,    several) substitutions at positions corresponding to G23S, D27N,    A40I, V60E,K, N101D, G163S, Y220F, and T244E.-   14. The variant of any of paragraphs 1-13, wherein the variant    comprises substitutions at positions corresponding to    F51I,L+E56R+D57N+K98I+R118F+G163S+T231R+N233R+P256T and one or more    (e.g., several) substitutions at positions corresponding to G23S,    D27N, A40I, V60E,K, N101D, Y220F, and T244E.-   15. The variant of any of paragraphs 1-14, wherein the variant    comprises substitutions at positions corresponding to    F51I,L+E56R+D57N+K98I+R118F+G163S+T231R+N233R+T244E+P256T and one or    more (e.g., several) substitutions at positions corresponding to    G23S, D27N, A40I, V60E,K, N101D, and Y220F.-   16. The variant of any of paragraphs 1-15, wherein the variant    comprises substitutions at positions corresponding to    F51I,L+E56R+D57N+V60E,K+K98I+R118F+T231R+N233R+P256T and one or more    (e.g., several) substitutions at positions corresponding to G23S,    D27N, A40I, N101D, G163S, Y220F, and T244E.-   17. The variant of any of paragraphs 1-16, wherein the variant    comprises substitutions at positions corresponding to    F51I,L+E56R+D57N+N101D+K98I+R118F+T231R+N233R+P256T and one or more    (e.g., several) substitutions at positions corresponding to G23S,    D27N, A40I, V60E,K, N101D, G163S, Y220F, and T244E.-   18. The variant of any of paragraphs 1-17, wherein the variant    comprises substitutions at positions corresponding to    F51I,L+E56R+D57N+V60E,K+K98I+N101D+R118F+T231R+N233R+P256T and one    or more (e.g., several) substitutions at positions corresponding to    G23S, D27N, A40I, G163S, Y220F, and T244E.-   19. The variant of any of paragraphs 1-18, wherein the variant    comprises substitutions at positions corresponding to one of the    following set of substitutions:-   E56R+R118F+T231R+N233R;-   R118F+T231R+N233R+P256T;-   A40I+R118F+T231R+N233R;-   F51I+E56R+R118F+T231R+N233R;-   F51L+E56R+R118F+T231R+N233R;-   E56R+D57N+R118F+T231R+N233R;-   E56R+V60K+R118F+T231R+N233R;-   G23S+E56R+R118F+T231R+N233R;-   D27N+E56R+R118F+T231R+N233R;-   F51I+E56R+R118F+T231R+N233R;-   E56R+R118F+T231R+N233R+P256T;-   G23S+D27N+E56R+R118F+T231R+N233R;-   G23S+F51I+E56R+R118F+T231R+N233R;-   G23S+E56R+R118F+T231R+N233R+P256T;-   D27N+F51I+E56R+R118F+T231R+N233R;-   D27N+E56R+R118F+T231R+N233R+P256T;-   F51I+E56R+R118F+T231R+N233R+P256T;-   G23S+D27N+F51I+E56R+R118F+T231R+N233R;-   G23S+D27N+E56R+R118F+T231R+N233R+P256T;-   G23S+D27N+F51I+E56R+V60K+R118F+T231R+N233R+P256T;-   G23S+D27N+F51I+E56R+V60E+R118F+T231R+N233R+P256T;-   G23S+F51I+E56R+R118F+T231R+N233R+P256T;-   D27N+F51I+E56R+R118F+T231R+N233R+P256T;-   G23S+D27N+F51I+E56R+R118F+T231R+N233R+P256T;-   A40I+E56R+R118F+T231R+N233R;-   F51L+E56R+R118F+T231R+N233R;-   D57N+E56R+R118F+T231R+N233R;-   K98I+E56R+R118F+T231R+N233R;-   G163S+E56R+R118F+T231R+N233R;-   A40I+F51L+E56R+R118F+T231R+N233R;-   A40I+D57N+E56R+R118F+T231R+N233R;-   A40I+K98I+E56R+R118F+T231R+N233R;-   A40I+G163S+E56R+R118F+T231R+N233R;-   A40I+E56R+R118F+T231R+N233R+P256T;-   F51L+D57N+E56R+R118F+T231R+N233R;-   F51L+K98I+E56R+R118F+T231R+N233R;-   F51L+G163S+E56R+R118F+T231R+N233R;-   F51L+E56R+R118F+T231R+N233R+P256T;-   D57N+K98I+E56R+R118F+T231R+N233R;-   D57N+G163S+E56R+R118F+T231R+N233R;-   D57N+E56R+R118F+T231R+N233R+P256T;-   K98I+G163S+E56R+R118F+T231R+N233R;-   K98I+E56R+R118F+T231R+N233R+P256T;-   G163S+E56R+R118F+T231R+N233R+P256T;-   A40I+F51L+D57N+E56R+R118F+T231R+N233R;-   A40I+F51L+K98I+E56R+R118F+T231R+N233R;-   A40I+F51L+G163S+E56R+R118F+T231R+N233R;-   A40I+F51L+E56R+R118F+T231R+N233R+P256T;-   A40I+D57N+K98I+E56R+R118F+T231R+N233R;-   A40I+D57N+G163S+E56R+R118F+T231R+N233R;-   A40I+D57N+E56R+R118F+T231R+N233R+P256T;-   A40I+K98I+G163S+E56R+R118F+T231R+N233R;-   A40I+K98I+E56R+R118F+T231R+N233R+P256T;-   A40I+G163S+E56R+R118F+T231R+N233R+P256T;-   F51L+D57N+K98I+E56R+R118F+T231R+N233R;-   F51L+D57N+G163S+E56R+R118F+T231R+N233R;-   F51L+D57N+E56R+R118F+T231R+N233R+P256T;-   F51L+K98I+G163S+E56R+R118F+T231R+N233R;-   F51L+K98I+E56R+R118F+T231R+N233R+P256T;-   F51L+G163S+E56R+R118F+T231R+N233R+P256T;-   D57N+K98I+G163S+E56R+R118F+T231R+N233R;-   D57N+K98I+E56R+R118F+T231R+N233R+P256T;-   D57N+G163S+E56R+R118F+T231R+N233R+P256T;-   K98I+G163S+E56R+R118F+T231R+N233R+P256T;-   A40I+F51L+D57N+K98I+E56R+R118F+T231R+N233R;-   A40I+F51L+D57N+G163S+E56R+R118F+T231R+N233R;-   A40I+F51L+D57N+E56R+R118F+T231R+N233R+P256T;-   A40I+F51L+K98I+G163S+E56R+R118F+T231R+N233R;-   A40I+F51L+K98I+E56R+R118F+T231R+N233R+P256T;-   A40I+F51L+G163S+E56R+R118F+T231R+N233R+P256T;-   A40I+D57N+K98I+G163S+E56R+R118F+T231R+N233R;-   A40I+D57N+K98I+E56R+R118F+T231R+N233R+P256T;-   A40I+D57N+G163S+E56R+R118F+T231R+N233R+P256T;-   A40I+K98I+G163S+E56R+R118F+T231R+N233R+P256T;-   F51L+D57N+K98I+G163S+E56R+R118F+T231R+N233R;-   F51L+D57N+K98I+E56R+R118F+T231R+N233R+P256T;-   F51L+D57N+G163S+E56R+R118F+T231R+N233R+P256T;-   F51L+K98I+G163S+E56R+R118F+T231R+N233R+P256T;-   D57N+K98I+G163S+E56R+R118F+T231R+N233R+P256T;-   A40I+F51L+D57N+K98I+G163S+E56R+R118F+T231R+N233R;-   A40I+F51L+D57N+K98I+E56R+R118F+T231R+N233R+P256T;-   A40I+F51L+D57N+G163S+E56R+R118F+T231R+N233R+P256T;-   A40I+F51L+K98I+G163S+E56R+R118F+T231R+N233R+P256T;-   A40I+D57N+K98I+G163S+E56R+R118F+T231R+N233R+P256T;-   F51L+D57N+K98I+G163S+E56R+R118F+T231R+N233R+P256T;-   A40I+F51L+D57N+K98I+G163S+E56R+R118F+T231R+N233R+P256T;-   A40I+F51L+E56R+D57N+K98I+R118F+G163S+T231R+N233R+P256T;-   A40I+E56R+R118F+T231R+N233R;-   E56R+R118F+T231R+N233R+T244E;-   G23S+D27N+E56R+R118F+T231R+N233R;-   G23S+A40I+E56R+R118F+T231R+N233R;-   G23S+F51I+E56R+R118F+T231R+N233R;-   G23S+E56R+R118F+T231R+N233R+T244E;-   G23S+E56R+R118F+T231R+N233R+P256T;-   D27N+A40I+E56R+R118F+T231R+N233R;-   D27N+F51I+E56R+R118F+T231R+N233R;-   D27N+E56R+R118F+T231R+N233R+T244E;-   D27N+E56R+R118F+T231R+N233R+P256T;-   A40I+F51I+E56R+R118F+T231R+N233R;-   A40I+E56R+R118F+T231R+N233R+T244E;-   A40I+E56R+R118F+T231R+N233R+P256T;-   F51I+E56R+R118F+T231R+N233R+T244E;-   F51I+E56R+R118F+T231R+N233R+P256T;-   E56R+R118F+T231R+N233R+T244E+P256T;-   G23S+D27N+A40I+E56R+R118F+T231R+N233R;-   G23S+D27N+A40I+E56R+V60K+R118F+T231R+N233R;-   G23S+D27N+A40I+E56R+V60E+R118F+T231R+N233R;-   G23S+D27N+F51I+E56R+R118F+T231R+N233R;-   G23S+D27N+E56R+R118F+T231R+N233R+T244E;-   G23S+D27N+E56R+R118F+T231R+N233R+P256T;-   G23S+A40I+F51I+E56R+R118F+T231R+N233R;-   G23S+A40I+E56R+R118F+T231R+N233R+T244E;-   G23S+A40I+E56R+R118F+T231R+N233R+P256T;-   G23S+F51I+E56R+R118F+T231R+N233R+T244E;-   G23S+F51I+E56R+R118F+T231R+N233R+P256T;-   G23S+E56R+R118F+T231R+N233R+T244E+P256T;-   G23S+E56R+V60K+R118F+T231R+N233R+T244E+P256T;-   G23S+E56R+V60E+R118F+T231R+N233R+T244E+P256T;-   D27N+A40I+F51I+E56R+R118F+T231R+N233R;-   D27N+A40I+E56R+R118F+T231R+N233R+T244E;-   D27N+A40I+E56R+R118F+T231R+N233R+P256T;-   D27N+F51I+E56R+R118F+T231R+N233R+T244E;-   D27N+F51I+E56R+R118F+T231R+N233R+P256T;-   D27N+E56R+R118F+T231R+N233R+T244E+P256T;-   A40I+F51I+E56R+R118F+T231R+N233R+T244E;-   A40I+F51I+E56R+R118F+T231R+N233R+P256T;-   A40I+E56R+R118F+T231R+N233R+T244E+P256T;-   F51I+E56R+R118F+T231R+N233R+T244E+P256T;-   F51I+E56R+V60K+R118F+T231R+N233R+T244E+P256T;-   F51I+E56R+V60E+R118F+T231R+N233R+T244E+P256T;-   G23S+D27N+A40I+F51I+E56R+R118F+T231R+N233R;-   G23S+D27N+A40I+F51I+E56R+V60K+R118F+T231R+N233R;-   G23S+D27N+A40I+F51I+E56R+V60E+R118F+T231R+N233R;-   G23S+D27N+A40I+E56R+R118F+T231R+N233R+T244E;-   G23S+D27N+A40I+E56R+V60K+R118F+T231R+N233R+T244E:-   G23S+D27N+A40I+E56R+V60E+R118F+T231R+N233R+T244E;-   G23S+D27N+A40I+E56R+R118F+T231R+N233R+P256T;-   G23S+D27N+F51I+E56R+R118F+T231R+N233R+T244E;-   G23S+D27N+F51I+E56R+R118F+T231R+N233R+P256T;-   G23S+D27N+E56R+R118F+T231R+N233R+T244E+P256T;-   G23S+A40I+F51I+E56R+R118F+T231R+N233R+T244E;-   G23S+A40I+F51I+E56R+R118F+T231R+N233R+P256T;-   G23S+A40I+E56R+R118F+T231R+N233R+T244E+P256T;-   G23S+F51I+E56R+R118F+T231R+N233R+T244E+P256T;-   D27N+A40I+F51I+E56R+R118F+T231R+N233R+T244E;-   D27N+A40I+F51I+E56R+R118F+T231R+N233R+P256T;-   D27N+A40I+E56R+R118F+T231R+N233R+T244E+P256T;-   D27N+F51I+E56R+R118F+T231R+N233R+T244E+P256T;-   A40I+F51I+E56R+R118F+T231R+N233R+T244E+P256T;-   A40I+F51I+E56R+V60K+R118F+T231R+N233R+T244E+P256T;-   A40I+F51I+E56R+V60E+R118F+T231R+N233R+T244E+P256T;-   G23S+D27N+A40I+F51I+E56R+R118F+T231R+N233R+T244E;-   G23S+D27N+A40I+F51I+E56R+V60K+R118F+T231R+N233R+T244E;-   G23S+D27N+A40I+F51I+E56R+R118F+T231R+N233R+P256T;-   G23S+D27N+A40I+F51I+E56R+V60K+R118F+T231R+N233R+P256T;-   G23S+D27N+A40I+F51I+E56R+V60E+R118F+T231R+N233R+P256T;-   G23S+D27N+A40I+E56R+R118F+T231R+N233R+T244E+P256T;-   G23S+D27N+F51I+E56R+R118F+T231R+N233R+T244E+P256T;-   G23S+A40I+F51I+E56R+R118F+T231R+N233R+T244E+P256T;-   D27N+A40I+F51I+E56R+R118F+T231R+N233R+T244E+P256T;-   D27N+A40I+F51I+E56R+V60K+R118F+T231R+N233R+T244E+P256T;-   D27N+A40I+F51I+E56R+V60E+R118F+T231R+N233R+T244E+P256T;-   G23S+D27N+A40I+F51I+E56R+R118F+T231R+N233R+T244E+P256T;-   G23S+D27N+A40I+F51I+E56R+K98I+N101D+R118F+T231R+N233R+T244E+P256T;-   G23S+D27N+A40I+F51I+E56R+V60K+R118F+T231R+N233R+T244E+P256T;-   G23S+D27N+A40I+F51I+E56R+V60E+R118F+T231R+N233R+T244E+P256T;-   F51I+E56R+R118F+T231R+N233R;-   E56R+R118F+T231R+N233R+T244E;-   D27N+F51I+E56R+R118F+T231R+N233R;-   D27N+E56R+R118F+T231R+N233R+T244E;-   F51I+E56R+R118F+T231R+N233R+T244E;-   D27N+F51I+E56R+R118F+T231R+N233R+T244E;-   G23S+E56R+R118F+T231R+N233R;-   D27N+E56R+R118F+T231R+N233R;-   K98I+E56R+R118F+T231R+N233R;-   Y220F+E56R+R118F+T231R+N233R;-   E56R+R118F+T231R+N233R+T244E;-   G23S+D27N+E56R+R118F+T231R+N233R;-   G23S+F51I+E56R+R118F+T231R+N233R;-   G23S+K98I+E56R+R118F+T231R+N233R;-   G23S+Y220F+E56R+R118F+T231R+N233R;-   G23S+E56R+R118F+T231R+N233R+T244E;-   G23S+E56R+R118F+T231R+N233R+P256T;-   D27N+F51I+E56R+R118F+T231R+N233R;-   D27N+K98I+E56R+R118F+T231R+N233R;-   D27N+Y220F+E56R+R118F+T231R+N233R;-   D27N+E56R+R118F+T231R+N233R+T244E;-   D27N+E56R+R118F+T231R+N233R+P256T;-   F51I+K98I+E56R+R118F+T231R+N233R;-   F51I+Y220F+E56R+R118F+T231R+N233R;-   F51I+E56R+R118F+T231R+N233R+T244E;-   F51I+E56R+R118F+T231R+N233R+P256T;-   K98I+Y220F+E56R+R118F+T231R+N233R;-   K98I+E56R+R118F+T231R+N233R+T244E;-   K98I+E56R+R118F+T231R+N233R+P256T;-   Y220F+E56R+R118F+T231R+N233R+T244E;-   Y220F+E56R+R118F+T231R+N233R+P256T;-   E56R+R118F+T231R+N233R+T244E+P256T;-   G23S+D27N+F51I+E56R+R118F+T231R+N233R;-   G23S+D27N+K98I+E56R+R118F+T231R+N233R;-   G23S+D27N+Y220F+E56R+R118F+T231R+N233R;-   G23S+D27N+E56R+R118F+T231R+N233R+T244E;-   G23S+D27N+E56R+R118F+T231R+N233R+P256T;-   G23S+F51I+K98I+E56R+R118F+T231R+N233R;-   G23S+F51I+Y220F+E56R+R118F+T231R+N233R;-   G23S+F51I+E56R+R118F+T231R+N233R+T244E;-   G23S+F51I+E56R+R118F+T231R+N233R+P256T;-   G23S+K98I+Y220F+E56R+R118F+T231R+N233R;-   G23S+K98I+E56R+R118F+T231R+N233R+T244E;-   G23S+K98I+E56R+R118F+T231R+N233R+P256T;-   G23S+Y220F+E56R+R118F+T231R+N233R+T244E;-   G23S+Y220F+E56R+R118F+T231R+N233R+P256T;-   G23S+E56R+R118F+T231R+N233R+T244E+P256T;-   D27N+F51I+K98I+E56R+R118F+T231R+N233R;-   D27N+F51I+Y220F+E56R+R118F+T231R+N233R;-   D27N+F51I+E56R+R118F+T231R+N233R+T244E;-   D27N+F51I+E56R+R118F+T231R+N233R+P256T;-   D27N+K98I+Y220F+E56R+R118F+T231R+N233R;-   D27N+K98I+E56R+R118F+T231R+N233R+T244E;-   D27N+K98I+E56R+R118F+T231R+N233R+P256T;-   D27N+Y220F+E56R+R118F+T231R+N233R+T244E;-   D27N+Y220F+E56R+R118F+T231R+N233R+P256T;-   D27N+E56R+R118F+T231R+N233R+T244E+P256T;-   F51I+K98I+Y220F+E56R+R118F+T231R+N233R;-   F51I+K98I+E56R+R118F+T231R+N233R+T244E;-   F51I+K98I+E56R+R118F+T231R+N233R+P256T;-   F51I+Y220F+E56R+R118F+T231R+N233R+T244E;-   F51I+Y220F+E56R+R118F+T231R+N233R+P256T;-   F51I+E56R+R118F+T231R+N233R+T244E+P256T;-   K98I+Y220F+E56R+R118F+T231R+N233R+T244E;-   K98I+Y220F+E56R+R118F+T231R+N233R+P256T;-   K98I+E56R+R118F+T231R+N233R+T244E+P256T;-   Y220F+E56R+R118F+T231R+N233R+T244E+P256T;-   G23S+D27N+F51I+K98I+E56R+R118F+T231R+N233R;-   G23S+D27N+F51I+Y220F+E56R+R118F+T231R+N233R;-   G23S+D27N+F51I+E56R+R118F+T231R+N233R+T244E;-   G23S+D27N+F51I+E56R+R118F+T231R+N233R+P256T;-   G23S+D27N+K98I+Y220F+E56R+R118F+T231R+N233R;-   G23S+D27N+K98I+E56R+R118F+T231R+N233R+T244E;-   G23S+D27N+K98I+E56R+R118F+T231R+N233R+P256T;-   G23S+D27N+Y220F+E56R+R118F+T231R+N233R+T244E;-   G23S+D27N+Y220F+E56R+R118F+T231R+N233R+P256T;-   G23S+D27N+E56R+R118F+T231R+N233R+T244E+P256T;-   G23S+F51I+K98I+Y220F+E56R+R118F+T231R+N233R;-   G23S+F51I+K98I+E56R+R118F+T231R+N233R+T244E;-   G23S+F51I+K98I+E56R+R118F+T231R+N233R+P256T;-   G23S+F51I+Y220F+E56R+R118F+T231R+N233R+T244E;-   G23S+F51I+Y220F+E56R+R118F+T231R+N233R+P256T;-   G23S+F51I+E56R+R118F+T231R+N233R+T244E+P256T;-   G23S+K98I+Y220F+E56R+R118F+T231R+N233R+T244E;-   G23S+K98I+Y220F+E56R+R118F+T231R+N233R+P256T;-   G23S+K98I+E56R+R118F+T231R+N233R+T244E+P256T;-   G23S+Y220F+E56R+R118F+T231R+N233R+T244E+P256T;-   D27N+F51I+K98I+Y220F+E56R+R118F+T231R+N233R;-   D27N+F51I+K98I+E56R+R118F+T231R+N233R+T244E;-   D27N+F51I+K98I+E56R+R118F+T231R+N233R+P256T;-   D27N+F51I+Y220F+E56R+R118F+T231R+N233R+T244E;-   D27N+F51I+Y220F+E56R+R118F+T231R+N233R+P256T;-   D27N+F51I+E56R+R118F+T231R+N233R+T244E+P256T;-   D27N+K98I+Y220F+E56R+R118F+T231R+N233R+T244E;-   D27N+K98I+Y220F+E56R+R118F+T231R+N233R+P256T;-   D27N+K98I+E56R+R118F+T231R+N233R+T244E+P256T;-   D27N+Y220F+E56R+R118F+T231R+N233R+T244E+P256T;-   F51I+K98I+Y220F+E56R+R118F+T231R+N233R+T244E;-   F51I+K98I+Y220F+E56R+R118F+T231R+N233R+P256T;-   F51I+K98I+E56R+R118F+T231R+N233R+T244E+P256T;-   F51I+Y220F+E56R+R118F+T231R+N233R+T244E+P256T;-   K98I+Y220F+E56R+R118F+T231R+N233R+T244E+P256T;-   G23S+D27N+F51I+K98I+Y220F+E56R+R118F+T231R+N233R;-   G23S+D27N+F51I+K98I+E56R+R118F+T231R+N233R+T244E;-   G23S+D27N+F51I+K98I+E56R+R118F+T231R+N233R+P256T;-   G23S+D27N+F51I+Y220F+E56R+R118F+T231R+N233R+T244E;-   G23S+D27N+F51I+Y220F+E56R+R118F+T231R+N233R+P256T;-   G23S+D27N+F51I+E56R+R118F+T231R+N233R+T244E+P256T;-   G23S+D27N+K98I+Y220F+E56R+R118F+T231R+N233R+T244E;-   G23S+D27N+K98I+Y220F+E56R+R118F+T231R+N233R+P256T;-   G23S+D27N+K98I+E56R+R118F+T231R+N233R+T244E+P256T;-   G23S+D27N+Y220F+E56R+R118F+T231R+N233R+T244E+P256T;-   G23S+F51I+K98I+Y220F+E56R+R118F+T231R+N233R+T244E;-   G23S+F51I+K98I+Y220F+E56R+R118F+T231R+N233R+P256T;-   G23S+F51I+K98I+E56R+R118F+T231R+N233R+T244E+P256T;-   G23S+F51I+Y220F+E56R+R118F+T231R+N233R+T244E+P256T;-   G23S+K98I+Y220F+E56R+R118F+T231R+N233R+T244E+P256T;-   D27N+F51I+K98I+Y220F+E56R+R118F+T231R+N233R+T244E;-   D27N+F51I+K98I+Y220F+E56R+R118F+T231R+N233R+P256T;-   D27N+F51I+K98I+E56R+R118F+T231R+N233R+T244E+P256T;-   D27N+F51I+Y220F+E56R+R118F+T231R+N233R+T244E+P256T;-   D27N+K98I+Y220F+E56R+R118F+T231R+N233R+T244E+P256T;-   F51I+K98I+Y220F+E56R+R118F+T231R+N233R+T244E+P256T;-   G23S+D27N+F51I+K98I+Y220F+E56R+R118F+T231R+N233R+T244E;-   G23S+D27N+F51I+K98I+Y220F+E56R+R118F+T231R+N233R+P256T;-   G23S+D27N+F51I+K98I+E56R+R118F+T231R+N233R+T244E+P256T;-   G23S+D27N+F51I+Y220F+E56R+R118F+T231R+N233R+T244E+P256T;-   G23S+D27N+K98I+Y220F+E56R+R118F+T231R+N233R+T244E+P256T;-   G23S+F51I+K98I+Y220F+E56R+R118F+T231R+N233R+T244E+P256T;-   D27N+F51I+K98I+Y220F+E56R+R118F+T231R+N233R+T244E+P256T;-   G23S+D27N+F51I+K98I+Y220F+E56R+R118F+T231R+N233R+T244E+P256T;-   G23S+D27N+F51I+E56R+K98I+R118F+Y220F+T231R+N233R+T244E+P256T.-   20. The variant of any of paragraphs 1-19, which is a variant of a    parent lipase selected from the group consisting of:

a) a polypeptide having at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99% or100% sequence identity to SEQ ID NO: 2;

b) a polypeptide encoded by a polynucleotide that hybridizes under lowstringency conditions, medium stringency conditions, medium-highstringency conditions, high stringency conditions, or very highstringency conditions with (i) the polypeptide coding sequence of SEQ IDNO: 1 or (ii) the full-length complement of (i);

c) a polypeptide encoded by a polynucleotide having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% sequence identity to SEQ ID NO: 1; and

d) a fragment of the polypeptide of SEQ ID NO: 2.

-   21. The variant of any of paragraphs 1-20, wherein the variant has    at least 60%, at least 65%, at least 70%, at least 75%, at least    80%, at least 85%, at least 90%, at least 95% identity, at least    96%, at least 97%, at least 98%, or at least 99%, but less than 100%    sequence identity to SEQ ID NO: 2.-   22. The variant of any of paragraphs 1-21, wherein the number of    substitutions is from 1-40, such as 1-30, such as 1-20, such as    1-12, such as 1-11, such as 1-10, such as 1-9, such as 1-8, such as    1-7, such as 1-6, such as 1-5, such as 1-4, such as 1-3, or 1, 2, 3    4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20    substitutions.-   23. The variant of any of paragraphs 1-22, wherein said variant in    comparison with the parent lipase has one or more of the following    properties: increased storage stability, increased thermostability,    improved wash performance and/or reduced odor generation.-   24. A composition comprising the variant of any of paragraphs 1-23.-   25. The composition of paragraph 25, further comprising a    surfactant.-   26. Use of the variant of any of paragraphs 1-23 for hydrolyzing a    lipase substrate.-   27. A method for cleaning a surface comprising contacting the    surface with the variant of any of paragraphs 1-23.-   28. A method of hydrolyzing a lipase substrate, comprising treating    the lipase substrate with a lipase variant of any of paragraphs    1-23.-   29. A polynucleotide encoding a variant of any of paragraphs 1-23.-   30. A nucleic acid construct comprising the polynucleotide of    paragraph 29, wherein the polynucleotide is operably linked to one    or more control sequences that direct the production of the lipase    variant in a recombinant host cell.-   31. An expression vector comprising the polynucleotide of paragraph    29 or nucleic acid construct of paragraph 30.-   32. A host cell comprising a nucleic acid construct of paragraph 30    or an expression vector of paragraph 31.-   33. A method of producing a lipase variant, comprising:

a) cultivating the host cell of paragraph 32 under conditions suitablefor expression of the variant; and

b) recovering the variant.

-   34. A microcapsule composition, wherein the membrane of the    microcapsule is produced by cross-linking of a polybranched    polyamine having a molecular weight of more than 1 kDa, wherein the    microcapsule comprising a lipase variant of any of paragraphs 1-23,-   35. The microcapsule composition of paragraph 34, further comprising    an enzyme is selected from the group consisting of protease,    amylase, lipase, cellulase, mannanase, pectinase, DNAse, laccase,    peroxidase, haloperoxidase, perhydrolase, and combinations thereof.-   36. The microcapsule composition of paragraph 34 or 35, wherein the    reactive amino groups of the polybranched polyamine constitute at    least 15% of the molecular weight.-   37. The microcapsule composition of any of paragraphs 34 to 36,    wherein the microcapsule is produced by using an acid chloride as    crosslinking agent.-   38. The microcapsule composition of any of paragraphs 34 to 37,    wherein the diameter of the microcapsule is at least, or above, 50    micrometers.-   39. The microcapsule composition of any of paragraphs 34 to 38,    wherein the microcapsule contains at least 1% by weight of active    enzyme, in particular lipase variant of any of paragraphs 1-23.-   40. The microcapsule composition of any of paragraphs 34 to 39,    which further includes an alcohol, such as a polyol.-   41. The microcapsule composition of any of paragraphs 34 to 40,    which contains less than 90% by weight of water.-   42. The microcapsule composition of paragraph 34 or 41, wherein the    protease is a metalloprotease or an alkaline serine protease, such    as a subtilisin.-   43. The microcapsule composition of any of paragraphs 34 to 42,    wherein the microcapsule is produced by interfacial polymerization    using an acid chloride as crosslinking agent.-   44. The microcapsule composition of any of paragraphs 34 to 43,    wherein the polybranched polyamine is a polyethyleneimine.-   45. The microcapsule composition of any of paragraphs 34 to 44,    wherein the microcapsule comprises a source of Mg2+, Ca2+, or Zn2+    ions, such as a poorly soluble salt of Mg2+, Ca2+, or Zn2+.-   46. A microcapsule composition, comprising a lipase variant of any    of paragraphs 1-23 entrapped in a compartment formed by a membrane,    which membrane is produced by cross-linking of (a) a polybranched    polyamine having a molecular weight of more than 800 Da, and (b) an    aliphatic or aromatic amine having a molecular weight of less than    300 Da; wherein the weight ratio of (a)/(b) is in the range of 0.1    to 1000.-   47. The microcapsule composition of paragraph 46, further comprising    an enzyme is selected from the group consisting of protease,    metalloprotease, subtilisin, amylase, lipase, cutinase, cellulase,    mannanase, pectinase, xanthanase, DNAse, laccase, peroxidase,    haloperoxidase, perhydrolase, and combinations thereof.-   48. The microcapsule composition of any of paragraphs 46 or 47,    wherein the reactive amino groups of the polybranched polyamine    constitute at least 15% of the molecular weight.-   49. The microcapsule composition of any of paragraphs 46 to 48,    wherein the diameter of the compartment is at least 50 micrometers.-   50. The microcapsule composition of any of paragraphs 46 to 49,    wherein the compartment contains at least 1% active enzyme by weight    of the total compartment, in particular lipase variant of any of    paragraphs 1-23.-   51. The microcapsule composition of any of paragraphs 46 to 50,    which further includes an alcohol, such as a polyol.-   52. The microcapsule composition of any of paragraphs 46 to 51,    wherein (a) is a polyethyleneimine.-   53. The composition of any of paragraphs 46 to 52, wherein (b) is an    ethyleneamine or alkanolamine.-   54. The microcapsule composition of any of paragraphs 46 to 5,    wherein (b) is selected from the group consisting of ethylene    diamine, diethylene triamine, triethylene tetraamine,    bis(3-aminopropyl)amine, monoethanolamine, diethanolamine,    triethanolamine, hexamethylene diamine, diamino benzene, piperazine,    and tetraethylene pentamine.-   55. The microcapsule composition of any of paragraphs 46 to 54,    wherein (b) is selected from the group consisting of diethylene    triamine, triethylene tetraamine, bis(3-aminopropyl)amine,    monoethanolamine, and diethanolamine.-   56. The microcapsule composition of any of paragraphs 46 to 55,    wherein the compartment comprises a source of Mg2+, Ca2+, or Zn2+    ions, such as a poorly soluble salt of Mg2+, Ca2+, or Zn2+.-   57. The microcapsule composition of any of paragraphs 43 to 55,    wherein the membrane is produced by using an acid chloride as    crosslinking agent, such as isophtaloyl chloride, terephthaloyl    chloride, or trimesoyl chloride.-   58. The microcapsule composition of any of paragraphs 46 to 57,    wherein the membrane is produced by interfacial polymerization.-   59. A liquid product comprising a microcapsule composition of any of    paragraphs 34-58.

The present invention is further described by the following examplesthat should not be construed as limiting the scope of the invention.

EXAMPLES Example 1 p-nitrophenyl (pNP) Assay

The hydrolytic activity of a lipase may be determined by a kinetic assayusing p-nitrophenyl acyl esters as substrate.

A 100 mM stock solution in DMSO for each of the substrates p-nitrophenylbutyrate (C4), p-nitrophenyl caproate (C6), p-nitrophenyl caprate (C10),p-nitrophenyl laurate (C12) and p-nitrophenyl palmitate (C16) (all fromSigma-Aldrich Danmark N S, Kirkebjerg Allé 84, 2605 Brøndby; Cat. no.:C3:N-9876, C6: N-0502, 010: N-0252, C12: N-2002, C16: N-2752) is dilutedto a final concentration of 1 mM 25 mM in the assay buffer (50 mM Tris;pH 7.7; 0.4% Triton X-100).

The lipase variants, the parent lipase and appropriate controls, e.g.,Lipolase™ (SEQ ID NO: 2) in 50 mM Hepes; pH 8.0; 10 ppm Triton X-100;+/−20 mM CaCl₂ are added to the substrate solution in the followingfinal protein concentrations: 0.01 mg/ml; 5×10⁻³ mg/ml; 2.5×10⁻⁴ mg/ml;and 1.25×10⁻⁴ mg/ml in 96-well NUNC plates (Cat. No. 260836, Kamstrupvej90, DK-4000, Roskilde). The buffer is also run as a negative control.Release of p-nitrophenol by hydrolysis of a p-nitrophenyl acyl may bemonitored at 405 nm for 5 minutes in 10 second intervals on a Spectramax 190 (Molecular Devices GmbH, Bismarckring 39, 88400 Biberach an derRiss, GERMANY). The hydrolytic activity towards one or more substratesof a variant may be compared to that of the parent lipase.

Example 2 Construction of Variants by Site-Directed Mutagenesis

Site-directed variants were constructed of the Thermomyces lanuginosuslipase (TLL) (SEQ ID NO: 2). The variants were made by traditionalcloning of DNA fragments (Sambrook et al., Molecular Cloning: ALaboratory Manual, 2nd Ed., Cold Spring Harbor, 1989) using PCR togetherwith properly designed mutagenic oligonucleotides that introduced thedesired mutations in the resulting sequence.

Mutagenic oligos were designed corresponding to the DNA sequenceflanking the desired site(s) of mutation, separated by the DNA basepairs defining the insertions/deletions/substitutions, and purchasedfrom an oligo vendor such as Life Technologies.

In order to test the TLL variants, the mutated DNA encoding a variantwasintegrated into a competent A. oryzae strain by homologousrecombination, fermented using standard protocols (yeast extract basedmedia, 3-4 days, 30° C.), and purified by chromatography. In thismanner, the variants listed in the Table below were constructed andproduced.

Variants of SEQ ID NO: 2

G23S + D27N + F51I + E56R + R118F + T231R + N233R + P256T A40I + F51L +E56R + D57N + K98I + R118F + G163S + T231R + N233R + P256T G23S + D27N +A40I + F51I + E56R + R118F + T231R + N233R + T244E + P256T D27N + F51I +E56R + R118F + T231R + N233R + T244E G23S + D27N + F51I + E56R + K98I +R118F + Y220F + T231R + N233R + T244E + P256T

Example 3 Relative Wash Performance (RP(Wash))

Washing experiments were performed using Automatic Mechanical StressAssay (AMSA) in order to assess the wash performance in laundry. TheAMSA plate has a number of slots for test solutions and a lid firmlysqueezing the laundry sample, the textile to be washed against all theslot openings. During the washing time, the plate, test solutions,textile and lid are vigorously shaken to bring the test solution incontact with the textile and apply mechanical stress in a regular,periodic oscillating manner. For further description see WO02/42740especially the paragraph “Special method embodiments” at page 23-24.

The laundry experiments were conducted under the experimental conditionsspecified below:

-   Detergent: 3.3 g/L Detergent B or 0.8 g/L Detergent J-   Test solution volume: 160 μL-   Wash time: 20 minutes-   Temperature: 30° C.-   Lipase dosage: 0 ppm or 0.35 ppm-   Test material: Cream Annatto stained EMPA221 cotton textile prepared    as described in WO06/125437 except to exchanging turmeric with    annatto (Annatto: A-320-WS, Chr. Hansen A/S, Boege Allé 10-12,    DK-2970, Hoersholm, Denmark & EMPA221: EMPA, Lerchenfeldstrasse 5,    CH-9014, St. Gallen, Switzerland)-   Water hardness was adjusted to 15° dH or 6° dH by addition of CaCl₂,    MgCl₂ and NaHCO₃ (Ca²⁺:Mg²⁺: HCO₃ ⁻=4:1:7.5 or 2:1:4.5).

TABLE 1 Composition: Model Detergent B (wt %) NaOH, pellets (>99%) 1.05Linear alkylbenzenesulfonic acid (LAS) (97%) 7.20 Sodium laureth sulfate(SLES) (28%) 10.58 Soy fatty acid (>90%) 2.75 Coco fatty acid (>99%)2.75 Alcohol ethoxylate (AEO) with 8 mol EO; 6.60 Lutensol TO 8 (~100%)Triethanol amine (100%) 3.33 Na-citrate, dihydrate (100%) 2.00 DTMPA;diethylenetriaminepentakis(meth- 0.48 ylene)pentakis(phosphonic acid),heptasodium salt (Dequest 2066 C) (~42% as Na7 salt) MPG (>98%) 6.00EtOH, propan-2-ol (90/10%) 3.00 Glycerol (>99.5%) 1.71 Sodium formate(>95%) 1.00 PCA (40% as sodium salt) 0.46 Water up to 100

TABLE 2 Composition: Model Detergent J (wt %) sodium hydroxide (>99%)0.50 Linear alkylbenzenesulfonic acid (LAS) (97%) 5.00 sodium alkylsulfate (90%) 5.00 AEOS, sodium (C12) alkyl ether sulfate (70.5%) 10.00Coco fatty acid (>99%) 1.00 Alcohol ethoxylate (AEO) with 7 mol EO(99.5%) 5.00 MEA, monoethanolamine (99.5%) 0.20 MPG (>98%) 3.00 EtOH,propan-2-ol (90/10%) 1.50 DTPA, diethylenetriaminepentaacetic acid, 0.10pentasodium salt, (~40% as Na5 salt) sodium citrate (>99%) 4.00 sodiumformate (>99%) 1.00 Water up to 100

After washing the textiles were flushed in tap water and excess waterwas removed from the textiles using filter paper and immediatelythereafter the textiles were dried at 100° C. for 15 minutes.

The wash performance was measured as the color change of the washedsoiled textile. The soil was cream mixed with annatto. Annatto containsthe colorant norbixin, which functions as a pH indicator with a pHdependent color change. Lipase activity leads to release of free fattyacids from the cream acylglycerols and this leads to pH decrease andthereby color change of the norbixin pH indicator. Lipase washperformance can therefore be expressed as the extent of color change oflight reflected-emitted from the washed soiled textile when illuminatedwith white light.

Color measurements were made with a professional flatbed scanner (EPSONEXPRESSION 11000XL, Atea NS, Lautrupvang 6, 2750 Ballerup, Denmark),which was used to capture an image of the washed soiled textile. Toextract a value for the light intensity from the scanned images, 24-bitpixel values from the image were converted into values for red, greenand blue (RGB).

Color change due to lipase activity was measured as the change in thereflection-emitting of green light (G) relative to the light intensityvalue (Int) calculated as:Int=√{square root over (R²+G²+B²)}

The relative wash performance (RP(Wash)) of a lipase relative to areference lipase was calculated as:RP(Wash)=(G/Int(tested lipase)−G/Int(no enzyme))/(G/Int(lipaseref.)−G/Int(no enzyme)).

A lipase is considered to exhibit improved wash performance, if itperforms better than the reference (RP(Wash)>1). In the context of thepresent invention the reference enzyme is the lipase shown as SEQ ID NO:2.

Odor Detection by Solid Phase Micro Extraction Gas ChromatographMeasurements.

The butyric acid release (odor) from the lipase washed swatches weremeasured by Solid Phase Micro Extraction Gas Chromatography (SPME-GC)using the following method.

Cream Annatto stained EMPA221 cotton textile was washed as specifiedabove and after wash, excess water was removed from the textile usingfilter paper and the textile was thereafter dried at 25° C. for 2 hours.Each SPME-GC measurement was performed with four pieces of the washedand dried textile (5 mm in diameter), which were transferred to a GasChromatograph (GC) vial and the vial was closed. The samples wereincubated at 30° C. for 24 hours and subsequently heated to 140° C. for30 minutes and stored at 20° C.-25° C. for at least 4 hours beforeanalysis. The analyses were performed on a Varian 3800 GC equipped witha Stabilwax-DA w/Integra-Guard column (30m, 0.32 mm ID and 0.25 micro-mdf) and a Carboxen PDMS SPME fiber (85 micro-m). Sampling from each GCvial was done at 50° C. for 8 minutes with the SPME fiber in thehead-space over the textile pieces and the sampled compounds weresubsequently injected onto the column (injector temperature=250° C.).Column flow=2 ml helium/minute. Column oven temperature gradient: 0minute=50° C., 2 minutes=50° C., 6 minutes 45 seconds=240° C., 11minutes 45 seconds=240° C. Detection was done using a Flame IonizationDetector (FID) and the retention time for butyric acid was identifiedusing an authentic standard.

The relative odor release (RP(Odor)) of a lipase is the ratio betweenthe amount butyric acid released (peak area) from a lipase washed swatchand the amount butyric acid released (peak area) from a reference lipasewashed swatch, after both values have been corrected for the amount ofbutyric acid released (peak area) from a non-lipase washed swatch(blank). The relative odor performance (RP(Odor)) of the polypeptide iscalculated in accordance with the below formula:RP(Odor)=(odor(tested lipase)−odor(no enzyme))/(odor(lipaseref.)−odor(no enzyme))Where odor is the measured butyric acid (peak area) released from thetextile surface.Benefit Risk factor (RP(Wash)/RP(Odor)).

The Benefit Risk factor (BRF) describing the wash performance (Benefit)compared to the odor release (Risk) can be defined as RP(Wash)/RP(Odor).If the Benefit Risk factor of a lipase is higher than 1, the lipase hasbetter wash performance relative to the released odor compared to thereference lipase (SEQ ID NO: 2).

AMSA AMSA AMSA AMSA Model J Model B Model B Model J RP(Wash) RP(Wash)RP(Wash) RP(6° dH, AMSA 6° dH, 0.8 6° dH, 3.3 15° dH, 3.3 0.8 g/L) ModelJ Construct Protein Mutations g/L) g/L) g/L) (odor) BRF SEQ ID NO: 21.00 1.00 1.00 1.00 1.00 G23S + D27N + F51I + E56R + R118F + 6.88 2.791.41 3.97 1.74 T231R + N233R + P256T A40I + F51L + E56R + D57N + 3.021.84 1.23 2.79 1.08 K98I + R118F + G163S + T231R + N233R + P256T G23S +D27N + A40I + F51I + 1.55 1.78 1.19 2.84 0.55 E56R + R118F + T231R +N233R + T244E + P256T D27N + F51I + E56R + 2.00 1.58 1.25 2.51 0.79R118F + T231R + N233R + T244E G23S + D27N + F51I + E56R + K98I + 2.801.72 1.30 2.41 1.16 R118F + Y220F + T231R + N233R + T244E + P256T

Example 4 Protein Thermal Unfolding Analysis (TSA, Thermal Shift Assay)

Protein thermal unfolding of variants of SEQ ID NO: 2 was monitored withSypro Orange (Invitrogen, S-6650) using a real-time PCR instrument(Applied Biosystems; Step-One-Plus).

In a 96-well white PCR-plate, 15 μl sample (purified enzyme diluted in100 mM EPPS, 0,01% Troton-X-100; pH8,0) was mixed (1:1) with SyproOrange (Conc.=10×; stock solution from supplier=5000×) in water.

The plate was sealed with an optical PCR seal. The PCR instrument wasset at a scan-rate of 76° C. per hour, starting at 25° C. and finishingat 96° C.

Fluorescence was monitored every 20 seconds using in-built LED bluelight for excitation and ROX-filter (610 nm, emission). Tm-values werecalculated as the maximum value of the first derivative (dF/dK) (Gregoryet al., 2009, J. Biomol. Screen. 14: 700).

Tm Construct Protein Mutations (° C.) SEQ ID NO: 1 74.7 G23S + D27N +F51I + E56R + R118F + T231R + N233R + 78.3 P256T A40I + F51L + E56R +D57N + K98I + R118F + G163S + 78.8 T231R + N233R + P256T G23S + D27N +A40I + F51I + E56R + R118F + T231R + 81.0 N233R + T244E + P256T D27N +F51I + E56R + R118F + T231R + N233R + T244E 76.4 G23S + D27N + F51I +E56R + K98I + R118F + Y220F + 77.7 T231R + N233R + T244E + P256T

Example 5 Construction of Variants by Site-Directed Mutagenesis

Site-directed variants were constructed of the Thermomyces lanuginosuslipase (TLL) (SEQ ID NO: 2). The variants were made by traditionalcloning of DNA fragments (Sambrook et al., Molecular Cloning: ALaboratory Manual, 2nd Ed., Cold Spring Harbor, 1989) using PCR togetherwith properly designed mutagenic oligonucleotides that introduced thedesired mutations in the resulting sequence.

Mutagenic oligos were designed corresponding to the DNA sequenceflanking the desired site(s) of mutation, separated by the DNA basepairs defining the insertions/deletions/substitutions, and purchasedfrom an oligo vendor such as Life Technologies. In order to test the TLLvariants, the mutated DNA encoding a variant was integrated into acompetent A. oryzae strain by homologous recombination, fermented usingstandard protocols (yeast extract based media, 3-4 days, 30° C.), andpurified by chromatography. In this manner, the variants listed in theTable below were constructed and produced.

Variants of SEQ ID NO: 2 G23S + T231R + N233R D27N + T231R + N233RA40I + T231R + N233R F51I + T231R + N233R F51L + T231R + N233R E56R +T231R + N233R D57N + T231R + N233R V60E + T231R + N233R V60K + T231R +N233R K98I + T231R + N233R N101D + T231R + N233R R118F + T231R + N233RG163S + T231R + N233R Y220F + T231R + N233R T231R + N233R + T244ET231R + N233R + P256T E56R + R118F + T231R + N233R R118F + T231R +N233R + P256T A40I + R118F + T231R + N233R F51I + E56R + R118F + T231R +N233R F51L + E56R + R118F + T231R + N233R E56R + D57N + R118F + T231R +N233R E56R + V60K + R118F + T231R + N233R G23S + D27N + F51I + E56R +V60E + R118F + T231R + N233R + P256T G23S + D27N + A40I + F51I + E56R +K98I + N101D + R118F + T231R + N233R + T244E + P256T G23S + D27N +A40I + F51I + E56R + V60K + R118F + T231R + N233R + T244E + P256T

Example 6 Relative Wash Performance (RP(Wash))

Washing experiments were performed using Automatic Mechanical StressAssay (AMSA) in order to assess the wash performance in laundry. TheAMSA plate has a number of slots for test solutions and a lid firmlysqueezing the laundry sample, the textile to be washed against all theslot openings. During the washing time, the plate, test solutions,textile and lid are vigorously shaken to bring the test solution incontact with the textile and apply mechanical stress in a regular,periodic oscillating manner. For further description see WO 02/42740especially the paragraph “Special method embodiments” at page 23-24.

The laundry experiments were conducted under the experimental conditionsspecified below:

-   Detergent: 3.3 g/L Model Detergent B or 0.8 g/L Model Detergent J-   Test solution volume: 160 μL-   Wash time: 20 minutes-   Temperature: 30° C.-   Lipase dosage: 0 ppm or 0.35 ppm-   Test material: Cream Annatto stained EMPA221 cotton textile prepared    as described in WO 06/125437 except to exchanging turmeric with    annatto (Annatto: A-320-WS, Chr. Hansen A/S, Boege Allé 10-12,    DK-2970, Hoersholm, Denmark & EMPA221: EMPA, Lerchenfeldstrasse 5,    CH-9014, St. Gallen, Switzerland)-   Water hardness was adjusted to 15° dH or 6° dH by addition of CaCl₂,    MgCl₂ and NaHCO₃ (Ca²⁺:Mg²⁺: HCO₃ ⁻=4:1:7.5 or 2:1:4.5).

TABLE 3 Composition: Model Detergent B (wt %) NaOH, pellets (>99%) 1.05Linear alkylbenzenesulfonic acid (LAS) (97%) 7.20 Sodium laureth sulfate(SLES) (28%) 10.58 Soy fatty acid (>90%) 2.75 Coco fatty acid (>99%)2.75 Alcohol ethoxylate (AEO) with 8 mol EO; 6.60 Lutensol TO 8 (~100%)Triethanol amine (100%) 3.33 Na-citrate, dihydrate (100%) 2.00 DTMPA;diethylenetriaminepentakis(meth- 0.48 ylene)pentakis(phosphonic acid),heptasodium salt (Dequest 2066 C) (~42% as Na7 salt) MPG (>98%) 6.00EtOH, propan-2-ol (90/10%) 3.00 Glycerol (>99.5%) 1.71 Sodium formate(>95%) 1.00 PCA (40% as sodium salt) 0.46 Water up to 100

TABLE 4 Composition: Model Detergent J (wt %) sodium hydroxide (>99%)0.50 Linear alkylbenzenesulfonic acid (LAS) (97%) 5.00 sodium alkylsulfate (90%) 5.00 AEOS, sodium (C12) alkyl ether sulfate (70.5%) 10.00Coco fatty acid (>99%) 1.00 Alcohol ethoxylate (AEO) with 7 mol EO(99.5%) 5.00 MEA, monoethanolamine (99.5%) 0.20 MPG (>98%) 3.00 EtOH,propan-2-ol (90/10%) 1.50 DTPA, diethylenetriaminepentaacetic acid, 0.10pentasodium salt, (~40% as Na5 salt) sodium citrate (>99%) 4.00 sodiumformate (>99%) 1.00 Water up to 100

After washing the textiles were flushed in tap water and excess waterwas removed from the textiles using filter paper and immediatelythereafter the textiles were dried at 100° C. for 15 minutes.

The wash performance was measured as the color change of the washedsoiled textile. The soil was cream mixed with annatto. Annatto containsthe colorant norbixin, which functions as a pH indicator with a pHdependent color change. Lipase activity leads to release of free fattyacids from the cream acylglycerols and this leads to pH decrease andthereby color change of the norbixin pH indicator. Lipase washperformance can therefore be expressed as the extent of color change oflight reflected-emitted from the washed soiled textile when illuminatedwith white light.

Color measurements were made with a professional flatbed scanner (EPSONEXPRESSION 11000XL, Atea NS, Lautrupvang 6, 2750 Ballerup, Denmark),which was used to capture an image of the washed soiled textile. Toextract a value for the light intensity from the scanned images, 24-bitpixel values from the image were converted into values for red, greenand blue (RGB).

Color change due to lipase activity was measured as the change in thereflection-emitting of green light (G) relative to the light intensityvalue (Int) calculated as:Int=√{square root over (R₂+G₂+B₂)}

The relative wash performance (RP(Wash)) of a lipase relative to areference lipase was calculated as:RP(Wash)=(G/Int(tested lipase)−G/Int(no enzyme))/(G/Int(lipaseref.)−G/Int(no enzyme)).

A lipase is considered to exhibit improved wash performance, if itperforms better than the reference (RP(Wash)>1). In the context of thepresent invention the reference enzyme is a lipase shown as SEQ ID NO:2.

Odor Detection by Solid Phase Micro Extraction Gas ChromatographMeasurements.

The butyric acid release (odor) from the lipase washed swatches weremeasured by Solid Phase Micro Extraction Gas Chromatography (SPME-GC)using the following method. Cream Annatto stained EMPA221 cotton textilewas washed as specified above and after wash, excess water was removedfrom the textile using filter paper and the textile was thereafter driedat 25° C. for 2 hours. Each SPME-GC measurement was performed with fourpieces of the washed and dried textile (5 mm in diameter), which weretransferred to a Gas Chromatograph (GC) vial and the vial was closed.The samples were incubated at 30° C. for 24 hours and subsequentlyheated to 140° C. for 30 minutes and stored at 20° C.-25° C. for atleast 4 hours before analysis. The analyses were performed on a Varian3800 GC equipped with a Stabilwax-DA w/Integra-Guard column (30 m, 0.32mm ID and 0.25 micro-m df) and a Carboxen PDMS SPME fiber (85 micro-m).Sampling from each GC vial was done at 50° C. for 8 minutes with theSPME fiber in the head-space over the textile pieces and the sampledcompounds were subsequently injected onto the column (injectortemperature=250° C.). Column flow=2 ml helium/minute. Column oventemperature gradient: 0 minute=50° C., 2 minutes=50° C., 6 minutes 45seconds=240° C., 11 minutes 45 seconds=240° C. Detection was done usinga Flame Ionization Detector (FID) and the retention time for butyricacid was identified using an authentic standard.

The relative odor release (RP(Odor)) of a lipase is the ratio betweenthe amount butyric acid released (peak area) from a lipase washed swatchand the amount butyric acid released (peak area) from a reference lipasewashed swatch, after both values have been corrected for the amount ofbutyric acid released (peak area) from a non-lipase washed swatch(blank). The relative odor performance (RP(Odor)) is calculated inaccordance with the below formula:RP(Odor)=(odor(tested lipase)−odor(no enzyme))/(odor(lipaseref.)−odor(no enzyme))Where odor is the measured butyric acid (peak area) released from thetextile surface.Benefit Risk factor (RP(Wash)/RP(Odor)).

The Benefit Risk factor (BRF) describing the wash performance (Benefit)compared to the odor release (Risk) can be defined as RP(Wash)/RP(Odor).If the Benefit Risk factor of a lipase is higher than 1, the lipase hasbetter wash performance relative to the released odor compared to thereference lipase (SEQ ID NO: 2).

AMSA AMSA AMSA AMSA Model J Model J Model B Model B RP(6° dH, RP(6° dH,RP(6° dH, RP(15° dH, 0.8 g/L) Construct Protein Mutations 0.8 g/L) 3.3g/L) 3.3 g/L) (odor) SEQ ID NO: 2 1.00 1.00 1.00 1.00 T231R + N233R 5.502.06 4.83 11.00 G23S + D27N + F51I + E56R + V60E + 2.93 1.22 1.46 2.41R118F + T231R + N233R + P256T G23S + D27N + A40I + F51I + E56R + 1.051.38 1.26 1.78 K98I + N101D + R118F + T231R + N233R + T244E + P256TG23S + D27N + A40I + F51I + E56R + 2.66 1.43 1.79 3.18 V60K + R118F +T231R + N233R + T244E + P256T G23S + T231R + N233R 3.73 1.65 2.68 6.63D27N + T231R + N233R 3.97 1.72 4.09 5.08 A40I + T231R + N233R 4.83 1.943.52 5.16 F51I + T231R + N233R 4.82 1.94 3.80 6.45 F51L + T231R + N233R4.17 1.81 3.15 6.76 E56R + T231R + N233R 3.75 1.56 2.59 5.52 D57N +T231R + N233R 3.90 1.37 2.46 5.42 V60E + T231R + N233R 1.87 1.46 1.335.76 V60K + T231R + N233R 2.56 0.96 −0.38 5.20 K98I + T231R + N233R 4.802.05 4.56 3.99 N101D + T231R + N233R 4.34 2.07 3.21 4.65 R118F + T231R +N233R 4.30 2.10 3.24 10.12 G163S + T231R + N233R 3.30 1.61 2.06 6.60Y220F + T231R + N233R 2.76 0.93 0.42 4.36 T231R + N233R + T244E 2.551.04 0.08 5.21 T231R + N233R + P256T 7.82 2.38 5.71 7.76 E56R + R118F +T231R + N233R 4.96 1.77 2.97 13.97 R118F + T231R + N233R + P256T 5.101.89 2.68 12.22 A40I + R118F + T231R + N233R 2.11 1.22 1.06 5.68 F51I +E56R + R118F + T231R + N233R 2.69 1.64 0.36 7.59 F51L + E56R + R118F +T231R + N233R 3.32 1.67 1.52 7.46 E56R + D57N + R118F + T231R + N233R2.96 1.31 0.75 9.75 E56R + V60K + R118F + T231R + N233R 3.14 1.21 1.2313.73

Example 7 Protein Thermal Unfolding Analysis (TSA, Thermal Shift Assay)

Protein thermal unfolding of variants of SEQ ID NO: 2 was monitored withSypro Orange (Invitrogen, S-6650) using a real-time PCR instrument(Applied Biosystems; Step-One-Plus). In a 96-well white PCR-plate, 15 μlsample (purified enzyme diluted in 100 mM EPPS, 0,01% Troton-X-100;pH8,0) was mixed (1:1) with Sypro Orange (Conc.=10×; stock solution fromsupplier=5000×) in water.

The plate was sealed with an optical PCR seal. The PCR instrument wasset at a scan-rate of 76° C. per hour, starting at 25° C. and finishingat 96° C.

Fluorescence was monitored every 20 seconds using in-built LED bluelight for excitation and ROX-filter (610 nm, emission). Tm-values werecalculated as the maximum value of the first derivative (dF/dK) (Gregoryet al., 2009, J. Biomol. Screen. 14: 700).

Tm Construct Protein Mutations (° C.) SEQ ID NO: 2 72.6 T231R + N233R71.5 G23S + D27N + F51I + E56R + V60E + R118F + 77.3 T231R + N233R +P256T G23S + D27N + A40I + F51I + E56R + K98I + N101D + 79.8 R118F +T231R + N233R + T244E + P256T G23S + D27N + A40I + F51I + E56R + V60K +80.0 R118F + T231R + N233R + T244E + P256T G23S + T231R + N233R 72.6D27N + T231R + N233R 72.4 A40I + T231R + N233R 74.0 F51I + T231R + N233R72.2 F51L + T231R + N233R 71.9 E56R + T231R + N233R 72.3 D57N + T231R +N233R 72.1 V60E + T231R + N233R 71.1 V60K + T231R + N233R 71.6 K98I +T231R + N233R 72.0 N101D + T231R + N233R 72.3 R118F + T231R + N233R 72.7G163S + T231R + N233R 71.9 Y220F + T231R + N233R 70.0 T231R + N233R +T244E 72.3 T231R + N233R + P256T 75.2 E56R + R118F + T231R + N233R 73.1R118F + T231R + N233R + P256T 75.7 A40I + R118F + T231R + N233R 74.6F51I + E56R + R118F + T231R + N233R 74.2 F51L + E56R + R118F + T231R +N233R 73.9 E56R + D57N + R118F + T231R + N233R 72.6 E56R + V60K +R118F + T231R + N233R 73.1

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

The invention claimed is:
 1. A variant of a parent lipase wherein saidvariant has lipase activity and has at least 90%, but less than 100%sequence identity with SEQ ID NO: 2, wherein said variant comprises asubstitution at positions corresponding to R118F, T231R, and N233R ofSEQ ID NO: 2 and one or more substitutions at positions corresponding toG23S, D27N, A401, F51I,L, E56R, D57N, V60E,K, K981, N101D, G163S, Y220F,T244E, and P256T of SEQ ID NO: 2, and said variant has improved washperformance compared to the lipase shown as SEQ ID NO:
 2. 2. The variantof claim 1, wherein the variant comprises substitutions corresponding toE56R+T231R+N233R of SEQ ID NO: 2 and one or more substitutions atpositions corresponding to G23S, D27N, A401, F511,L, D57N, V60E,K, K981,N101D, G163S, Y220F, T244E, and P256T of SEQ ID NO:
 2. 3. The variant ofclaim 1, wherein the variant comprises substitutions at positionscorresponding to E56R+R118F+T231R+N233R of SEQ ID NO: 2 and one or moresubstitutions at positions corresponding to G23S, D27N, A401, F511,L,D57N, V60E,K, K981, N101D, G163S, Y220F, T244E, and P256T of SEQ ID NO:2.
 4. The variant of claim 1, wherein the variant comprisessubstitutions at positions corresponding to E56R+R118F+T231R+N233R+P256Tof SEQ ID NO: 2 and one or more substitutions at positions correspondingto G23S, D27N, A401, F511,L, D57N, V60E,K, K981, N101D, G163S, Y220F,and T244E of SEQ ID NO:
 2. 5. The variant of claim 1, wherein thevariant comprises substitutions at positions corresponding toF51I,L+E56R+R118F+T231R+N233R+P256T of SEQ ID NO: 2 and one or moresubstitutions at positions corresponding to G23S, D27N, A401, D57N,V60E,K, K981, N101D, G163S, Y220F, and T244E of SEQ ID NO:
 2. 6. Thevariant of claim 1, wherein the variant comprises substitutions atpositions corresponding to G23S+F51I,L+E56R+R118F+T231R+N233R+P256T ofSEQ ID NO: 2 and one or more substitutions at positions corresponding toD27N, A401, D57N, V60E,K, K981, N101D, G163S, Y220F, and T244E of SEQ IDNO:
 2. 7. The variant of claim 1, wherein the variant comprisessubstitutions at positions corresponding toD27N+F51I,L+E56R+R118F+T231R+N233R+P256T of SEQ ID NO: 2 and one or moresubstitutions at positions corresponding to G23S, A401, D57N, V60E,K,K981, N101D, G163S, Y220F, and T244E of SEQ ID NO:
 2. 8. The variant ofclaim 1, wherein the variant comprises substitutions at positionscorresponding to A401+F511,L+E56R+R118F+T231R+N233R+P256T of SEQ ID NO:2 and one or more substitutions at positions corresponding to G23S,D27N, D57N, V60E,K, K981, N101D, G163S, Y220F, and T244E of SEQ ID NO:2.
 9. The variant of claim 1, wherein the variant comprisessubstitutions at positions corresponding toF51I,L+E56R+D57N+R118F+T231R+N233R+P256T of SEQ ID NO: 2 and one or moresubstitutions at positions corresponding to G23S, D27N, A401, V60E,K,K981, N101D, G163S, Y220F, and T244E of SEQ ID NO:
 2. 10. The variant ofclaim 1, wherein the variant comprises substitutions at positionscorresponding to F511,L+E56R+D57N+K981+R118F+T231R+N233R+P256T of SEQ IDNO: 2 and one or more substitutions at positions corresponding to G23S,D27N, A401, V60E,K, N101D, G163S, Y220F, and T244E of SEQ ID NO:
 2. 11.The variant of claim 1, wherein the variant comprises substitutions atpositions corresponding toF51I,L+E56R+D57N+K98I+R118F+G163S+T231R+N233R+P256T of SEQ ID NO: 2 andone or more substitutions at positions corresponding to G23S, D27N,A401, V60E,K, N101D, Y220F, and T244E of SEQ ID NO:
 2. 12. The variantof claim 1, wherein the variant comprises substitutions at positionscorresponding toF511,L+E56R+D57N+K981+R118F+G163S+T231R+N233R+T244E+P256T of SEQ ID NO:2 and one or more substitutions at positions corresponding to G23S,D27N, A401, V60E,K, N101D, and Y220F of SEQ ID NO:
 2. 13. The variant ofclaim 1, wherein the variant comprises substitutions at positionscorresponding to F51I, L+E56R+D57N+V60E, K+K981+R118F+T231R+N233R+P256Tof SEQ ID NO: 2 and one or more substitutions at positions correspondingto G23S, D27N, A401, N101D, G163S, Y220F, and T244E of SEQ ID NO:
 2. 14.The variant of claim 1, wherein the variant comprises substitutions atpositions corresponding toF51I,L+E56R+D57N+N101D+K98I+R118F+T231R+N233R+P256T of SEQ ID NO: 2 andone or more substitutions at positions corresponding to G23S, D27N,A401, V60E,K, N101D, G163S, Y220F, and T244E of SEQ ID NO:
 2. 15. Thevariant of claim 1, wherein the variant comprises substitutions atpositions corresponding toF511,L+E56R+D57N+V60E,K+K981+N101D+R118F+T231R+N233R+P256T of SEQ ID NO:2 and one or more substitutions at positions corresponding to G23S,D27N, A401, G163S, Y220F, and T244E of SEQ ID NO:
 2. 16. A compositioncomprising the variant of claim 1 and a surfactant.
 17. A method forcleaning a surface comprising contacting the surface with the variant ofclaim
 1. 18. A polynucleotide encoding a variant of claim
 1. 19. Anucleic acid construct comprising the polynucleotide of claim 18,wherein the polynucleotide is operably linked to one or more controlsequences that direct the production of the lipase variant in arecombinant host cell.
 20. A host cell comprising a nucleic acidconstruct of claim
 19. 21. A method of producing a lipase variant,comprising: a) cultivating the host cell of claim 20 under conditionssuitable for expression of the variant; and b) recovering the variant.22. The variant of claim 1, wherein the variant comprises substitutionsat positions corresponding toG23S+D27N+F51I,L+E56R+R118F+T231R+N233R+P256T of SEQ ID NO:
 2. 23. Thevariant of claim 1, wherein the variant comprises substitutions atpositions corresponding toG23S+D27N+A40I+F511,L+E56R+R118F+T231R+N233R+T244E+P256T of SEQ ID NO:2.
 24. The variant of claim 1, wherein the variant comprisessubstitutions at positions corresponding toG23S+D27N+A401+F511,L+E56R+V60E,K+R118F+T231R+N233R+T244E+P256T of SEQID NO:
 2. 25. The variant of claim 24, wherein the variant has at least93% sequence identity, but less than 100% sequence identity, to SEQ IDNO:
 2. 26. The variant of claim 24, wherein the variant has at least 95%sequence identity, but less than 100% sequence identity, to SEQ ID NO:2.